S. epidermidis antigens

ABSTRACT

Hyperimmune serum reactive antigens and fragments thereof are disclosed. In addition, methods for isolating such antigens and specific uses thereof, including the treatment of  S. epidermidis  infections, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/540,696 filed on Jul. 3, 2012, which is a divisional of U.S.application Ser. No. 12/625,053 filed on Nov. 24, 2009, which is adivisional of U.S. application Ser. No. 10/551,492 filed on Oct. 13,2006, which is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/EP2004/003398 filed Mar. 31, 2004,which claims priority to European Application No. 03450078.5 filed Mar.31, 2003. The above-referenced applications are incorporated herein byreference in their entirety.

BACKGROUND

The present invention relates to isolated nucleic acid molecules, whichencode antigens for Staphylococcus epidermidis, which are suitable foruse in preparation of pharmaceutical medicaments for the prevention andtreatment of bacterial infections caused by Staphylococcus epidermidis.

Staphylococci are opportunistic pathogens, which can cause illnesses,which range from minor infections to life threatening diseases. Of thelarge number of Staphylococci at least 3 are commonly associated withhuman disease: S. aureus, S. epidermidis and rarely S. saprophyticus(Crossley, K. B. and Archer G. L, eds. (1997). The Staphylococci inHuman Disease. Churchill Livingston Inc.) Staphylococcal infections areimposing an increasing threat in hospitals worldwide. The appearance anddisease causing capacity of Staphylococci are related to the widespreaduse of antibiotics, which induced and continue to induce multi-drugresistance. Both S. aureus and S. epidermidis have become resistant tomany commonly used antibiotics, most importantly to methicillin (MRSA)and vancomycin (VISA). Drug resistance is an increasingly importantpublic health concern, and soon many infections caused by staphylococcimay be untreatable by antibiotics. In addition to its adverse effect onpublic health, antimicrobial resistance contributes to higher healthcare costs, since treating resistant infections often requires the useof more toxic and more expensive drugs, and can result in longerhospital stays for infected patients.

Moreover, even with the help of effective antibiotics, the most seriousstaphylococcal infections have 30-50% mortality.

Every human being is colonized with S. epidermidis. The normal habitatsof S. epidermidis are the skin and the mucous membrane. Generally, theestablished flora of the nose prevents acquisition of new strains.However, colonization with other strains may occur when antibiotictreatment is given that leads to elimination of the susceptible carrierstrain. Because this situation occurs in the hospitals, patients maybecome colonized with resistant nosocomial Staphylococci.

Staphylococci become potentially pathogenic as soon as the naturalbalance between microorganisms and the immune system gets disturbed,when natural barriers (skin, mucous membrane) are breached. Thecoagulase-positive S. aureus is the most pathogenic staphylococcalspecies, feared by surgeons for a long time. Most frequently it causessurgical wound infections, and induces the formation of abscesses. S.epidermidis causes diseases mostly related to the presence of foreignbodies and the use of devices, such as catheter related infections,cerebrospinal fluid shunt infections, peritonitis in dialysed patients(mainly CAPD), endocarditis in individuals with prosthetic valves. Thisis exemplified in immunocompromised individuals such as oncologypatients and premature neonates in whom coagulase-negativestaphylococcal infections frequently occur in association with the useof intravascular device. The increase in incidence is related to theincreased used of these devices and increasing number ofimmuno-compromised patients.

The pathogenesis of staphylococci is multifactorial. In order toinitiate infection the pathogen has to gain access to the cells andtissues of the host, that is adhere. Since adherence is obviously acrucial step in the initiation of foreign body infections, S.epidermidis is equipped with a number of cell surface molecules, whichpromote adherence to foreign material and through that mechanismestablish infection in the host. A characteristic of many pathogenicstrains of S. epidermidis is the production of a slime resulting inbiofilm formation. The slime is predominantly a secreted teichoic acid,normally found in the cell wall of the staphylococci. This ability toform a biofilm on the surface of a prosthetic device is probably asignificant determinant of virulence for these bacteria, since thisprevents phagocytosis of the bacteria. A further means of staphylococcito cause damage to its host are the secreted products, such asenterotoxins, exotoxins, and tissue damaging enzymes. The toxins kill ormisguide immune cells, which are important in the host defence. Theseveral different types of toxins are responsible for most of thesymptoms during infections.

For all the above-mentioned reasons there remains a need for aneffective preventive and therapeutic treatment, but until today there isno effective preventive or therapeutic vaccine approved. It has beenshown that an antibody deficiency state contributes to staphylococcalpersistence, suggesting that anti-staphylococcal antibodies areimportant in host defence. Antibodies-added as passive immunisation orinduced by active vaccination—directed towards surface components couldboth, prevent bacterial adherence, neutralize toxins and promotephagocytosis. An effective vaccine offers great potential for patientsfacing elective surgery in general, and those receiving endovasculardevices, in particular. Moreover, patients suffering from chronicdiseases, which decrease immune responses or undergoing continuousambulatory peritoneal dialysis are likely to benefit from such avaccine.

A vaccine can contain a whole variety of different antigens. Examples ofantigens are wholekilled or attenuated organisms, subfractions of theseorganisms/tissues, proteins, or, in their most simple form, peptides.Antigens can also be recognized by the immune system in form ofglycosylated proteins or peptides and may also be or containpolysaccharides or lipids. Short peptides can be used since for examplecytotoxic T-cells (CTL) recognize antigens in form of short usually 8-11amino acids long peptides in conjunction with major histocompatibilitycomplex (MHC). B-cells can recognize linear epitopes as short as 4-5amino acids, as well as three-dimensional structures (conformationalepitopes). In order to obtain sustained, antigen-specific immuneresponses, adjuvants need to trigger immune cascades that involve allcells of the immune system necessary. Primarily, adjuvants are acting,but are not restricted in their mode of action, on so-called antigenpresenting cells (APCs). These cells usually first encounter theantigen(s) followed by presentation of processed or unmodified antigento immune effector cells. Intermediate cell types may also be involved.Only effector cells with the appropriate specificity are activated in aproductive immune response. The adjuvant may also locally retainantigens and co-injected other factors. In addition the adjuvant may actas a chemoattractant for other immune cells or may act locally and/orsystemically as a stimulating agent for the immune system.

Approaches to develop a vaccine have focused until today mainly on S.aureus {Shinefield, H. et al., 2002}. Therefore it would be of greatvalue to develop a vaccine targeting S. epidermidis or preferentiallyboth Staphylococci.

The present inventors have developed a method for identification,isolation and production of hyperimmune serum reactive antigens from aspecific pathogen, especially from Staphylococcus aureus andStaphylococcus epidermidis (WO 02/059148). Importantly for the presentinvention, the selection of sera for the identification of antigens fromS. epidermidis is different from that applied to the previous screens.

Individuals undergoing continuous peritoneal dialysis represent one ofthe most important groups of patients infected by S. epidermidis.Staphylococci preferentially infect patients with foreign bodies such asdialysis catheters. Peritoneal dialysis patients suffer from peritonitismainly caused by S. aureus and coagulase negative staphylococci,especially S. epidermidis. In order to identify antigens expressed by S.epidermidis in humans during peritonitis, human serum samples werecollected from patients undergoing peritoneal dialysis for an extendedperiod of time and suffered from peritonitis caused by S. epidermidiswithin the previous 12 months, and thus considered to be in the lateconvalescent phase of the disease. It has been firmly established thatpatients with serious staphylococcal diseases—such asperitonitis—develop antibodies, which sustain for up to a year.

The problem underlying the present invention was to provide means forthe development of medicaments such as vaccines against S. epidermidisinfection. More particularly, the problem was to provide an efficientand relevant set of nucleic acid molecules or hyperimmune serum reactiveantigens from S. epidermidis that can be used for the manufacture ofsaid medicaments.

Therefore, the present invention provides an isolated nucleic acidmolecule encoding a hyperimmune serum reactive antigen or a fragmentthereof comprising a nucleic acid sequence, which is selected from thegroup consisting of:

-   a) a nucleic acid molecule having at least 70% sequence identity to    a nucleic acid molecule selected from Seq ID No 1, 4, 6-9, 11-13,    15, 17, 19, 21, 25-26, 28-31.-   b) a nucleic acid molecule which is complementary to the nucleic    acid molecule of a),-   c) a nucleic acid molecule comprising at least 15 sequential bases    of the nucleic acid molecule of a) or b)-   d) a nucleic acid molecule which anneals under stringent    hybridisation conditions to the nucleic acid molecule of a), b), or    c)-   e) a nucleic acid molecule which, but for the degeneracy of the    genetic code, would hybridise to the nucleic acid molecule defined    in a), b), c) or d).

According to a preferred embodiment of the present invention thesequence identity is at least 80%, preferably at least 95%, especially100%.

Furthermore, the present invention provides an isolated nucleic acidmolecule encoding a hyperimmune serum reactive antigen or a fragmentthereof comprising a nucleic acid sequence selected from the groupconsisting of

-   a) a nucleic acid molecule having at least 96% sequence identity to    a nucleic acid molecule selected from Seq ID No 2-3, 5, 10, 14, 16,    18, 22-24, 27,-   b) a nucleic acid molecule which is complementary to the nucleic    acid molecule of a),-   c) a nucleic acid molecule comprising at least 15 sequential bases    of the nucleic acid molecule of a) or b)-   d) a nucleic acid molecule which anneals under stringent    hybridisation conditions to the nucleic acid molecule of a), b) or    c),-   e) a nucleic acid molecule which, but for the degeneracy of the    genetic code, would hybridise to the nucleic acid defined in a),    b), c) or d).

According to another aspect, the present invention provides an isolatednucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of

-   a) a nucleic acid molecule selected from Seq ID No 20.-   b) a nucleic acid molecule which is complementary to the nucleic    acid of a),-   c) a nucleic acid molecule which, but for the degeneracy of the    genetic code, would hybridise to the nucleic acid defined in a),    b), c) or d).

Preferably, the nucleic acid molecule is DNA or RNA.

According to a preferred embodiment of the present invention, thenucleic acid molecule is isolated from a genomic DNA, especially from aS. epidermidis genomic DNA.

According to the present invention a vector comprising a nucleic acidmolecule according to any of the present invention is provided.

In a preferred embodiment the vector is adapted for recombinantexpression of the hyperimmune serum reactive antigens or fragmentsthereof encoded by the nucleic acid molecule according to the presentinvention.

The present invention also provides a host cell comprising the vectoraccording to the present invention.

According to another aspect the present invention further provides ahyperimmune serum-reactive antigen comprising an amino acid sequencebeing encoded by a nucleic acid molecule according to the presentinvention.

In a preferred embodiment the amino acid sequence (polypeptide) isselected from the group consisting of Seq ID No 32, 35, 37-40, 42-44,46, 48, 50, 52, 56-57, 59-62.

In another preferred embodiment the amino acid sequence (polypeptide) isselected from the group consisting of Seq ID No 33-34, 36, 41, 45, 47,49, 53-55, 58.

In a further preferred embodiment the amino acid sequence (polypeptide)is selected from the group consisting of Seq ID No 51.

According to a further aspect the present invention provides fragmentsof hyperimmune serum-reactive antigens selected from the groupconsisting of peptides comprising amino acid sequences of column“predicted immunogenic aa” and “location of identified immunogenicregion” of Table 1; the serum reactive epitopes of Table 1, especiallypeptides comprising amino acids 6-28, 54-59, 135-147, 193-205, 274-279,284-291, 298-308, 342-347, 360-366, 380-386, 408-425, 437-446, 457-464,467-477, 504-510, 517-530, 535-543, 547-553, 562-569, 573-579, 592-600,602-613, 626-631, 638-668 and 396-449 of Seq ID No 32; 5-24, 101-108,111-117, 128-142, 170-184, 205-211, 252-267, 308-316, 329-337, 345-353,360-371, 375-389, 393-399, 413-419, 429-439, 446-456, 471-485, 495-507,541-556, 582-588, 592-602, 607-617, 622-628, 630-640 and 8-21 of Seq IDNo 33; 10-20, 23-33, 40-45, 59-65, 72-107, 113-119, 127-136, 151-161 and33-59 of Seq ID No 34; 4-16, 28-34, 39-61, 66-79, 100-113, 120-127,130-137, 142-148, 150-157, 192-201, 203-210, 228-239, 245-250, 256-266,268-278, 288-294, 312-322, 336-344, 346-358, 388-396, 399-413, 425-430,445-461, 464-470, 476-482, 486-492, 503-511, 520-527, 531-541, 551-558,566-572, 609-625, 635-642, 650-656, 683-689, 691-705, 734-741, 750-767,782-789, 802-808, 812-818, 837-844, 878-885, 907-917, 930-936 and913-933 of Seq ID No 35; 5-12, 20-27, 46-78, 85-92, 104-112, 121-132,150-167, 179-185, 200-213, 221-227, 240-264, 271-279, 282-290, 311-317and 177-206 of Seq ID No 36; 18-24, 31-40, 45-51, 89-97, 100-123,127-132, 139-153, 164-170, 184-194, 200-205, 215-238, 244-255, 257-270,272-280, 289-302, 312-318, 338-348, 356-367 and 132-152 of Seq ID No 37;7-16, 39-45, 73-83, 90-98, 118-124, 130-136, 194-204, 269-280, 320-327,373-381, 389-397, 403-408, 424-430, 436-441, 463-476, 487-499, 507-514,527-534, 540-550, 571-577, 593-599, 620-629, 641-647, 650-664, 697-703,708-717, 729-742, 773-790, 794-805, 821-828, 830-837, 839-851, 858-908,910-917, 938-947, 965-980, 1025-1033, 1050-1056, 1073-1081, 1084-1098,1106-1120, 1132-1140, 1164-1170, 1185-1194, 1201-1208, 1215-1224,1226-1234, 1267-1279, 1325-1331, 1356-1364, 1394-1411, 1426-1439,1445-1461, 1498-1504, 1556-1561, 1564-1573, 1613-1639, 1648-1655,1694-1714, 1748-1755, 1778-1785, 1808-1813, 1821-1827, 1829-1837,1846-1852, 1859-1865, 1874-1883, 1895-1900, 1908-1913, 1931-1937,1964-1981, 1995-2005, 2020-2033, 2040-2047, 2103-2109, 2118-2127,2138-2144, 2166-2175, 2180-2187, 2220-2225, 2237-2242, 2247-2253,2273-2281, 2286-2306, 2314-2320, 2323-2345, 2350-2355, 2371-2384,2415-2424, 2426-2431, 2452-2472, 2584-2589, 2610-2621, 2638-2655,2664-2670, 2681-2690, 2692-2714, 2724-2730 and 687-730 of Seq ID No 38;10-40, 53-59, 79-85, 98-104, 117-122, 130-136, 144-158, 169-175,180-185, 203-223, 232-237, 243-254, 295-301 and 254-292 of Seq ID No 39;28-50, 67-85, 93-115, 120-134, 144-179, 240-249, 328-340, 354-360,368-400, 402-417, 419-427, 429-445, 447-455, 463-468, 472-480, 485-500,502-510, 512-534, 537-546, 553-558, 582-594, 619-637, 645-654, 690-709,735-745, 749-756, 786-792, 275-316 and 378-401 of Seq ID No 40; 5-16,21-30, 33-40, 52-74, 101-108, 116-122, 164-182, 185-219, 256-261,273-279, 285-291, 297-304, 312-328, 331-338, 355-362, 364-371, 373-401,411-423 and 191-208 of Seq ID No 41; 34-55, 67-74, 85-93, 105-115,138-152, 161-171, 182-189, 197-205, 213-219, 232-239, 241-248, 250-263,272-277, 288-299 and 216-231 of Seq ID No 42; 21-27, 32-37, 43-51,67-74, 82-92, 94-100, 106-112, 140-149, 153-159, 164-182, 193-215,222-227, 260-267, 308-322, 330-340, 378-387, 396-403, 417-432, 435-441,448-465, 476-482, 488-498, 500-510 and 214-280 of Seq ID No 43; 4-21,29-52, 80-87, 104-123, 126-133, 141-157, 182-189, 194-202, 214-220,227-235, 242-252 and 33-108 of Seq ID No 44; 12-18, 20-27, 29-59, 64-72,84-90, 96-103, 109-121, 125-155, 164-177, 179-186, 188-201, 216-227,235-253, 259-274, 276-294, 296-310, 322-339, 341-348, 369-379, 398-403,409-421 and 76-96 of Seq ID No 45; 4-15, 24-41, 71-80, 104-111, 113-119,123-130, 139-149, 168-178, 187-200 and 4-45 of Seq ID No 46; 13-19,32-37, 44-56 and 1-14 of Seq ID No 47; 6-11, 16-35, 75-81, 95-100,126-139, 206-214, 225-233, 241-259, 268-276, 319-325, 339-360, 371-401,435-441, 452-459, 462-472, 491-503, 505-516, 549-556, 567-580, 590-595,612-622, 624-630, 642-648, 656-662, 687-693, 698-704, 706-712, 736-750,768-777, 784-789, 812-818, 847-858, 894-900, 922-931, 938-949, 967-984,986-992, 1027-1032, 1041-1054, 1082-1088, 1091-1097, 1119-1124,1234-1240, 1250-1258, 1274-1289, 1299-1305, 1392-1398, 1400-1405,1429-1442, 1460-1474, 1505-1514, 1531-1537, 1540-1552, 1558-1571,1582-1587, 1616-1623, 1659-1666, 1671-1677, 1680-1686, 1698-1704,1706-1712, 1768-1774, 1783-1797, 1814-1819, 1849-1855, 1870-1876,1890-1897, 1947-1953, 1972-1980, 1999-2013, 2044-2051, 2068-2084,2093-2099, 2122-2131, 2142-2147, 2156-2163, 2170-2179, 2214-2220,2235-2245, 2271-2281, 2287-2293, 2308-2317, 2352-2362, 2373-2378,2387-2407, 2442-2448, 2458-2474, 2507-2516, 2531-2537, 2540-2551,2555-2561, 2586-2599, 2617-2627, 2644-2649, 2661-2675, 2685-2692,2695-2707, 2733-2739, 2741-2747, 2774-2783, 2788-2795, 2860-2870,2891-2903, 2938-2947, 2973-2980, 2993-2999, 3004-3030, 3046-3059,3066-3077, 3082-3088, 3120-3132, 3144-3149, 3153-3169, 3200-3212,3232-3256, 3276-3290, 3308-3322, 3330-3338, 3353-3360, 3363-3371,3390-3408, 3431-3447, 3454-3484, 3503-3515, 3524-3541, 3543-3550,3560-3567, 3586-3599, 3616-3621, 3642-3647, 3663-3679, 213-276, 579-621and 1516-1559 of Seq ID No 48; 19-41, 43-49, 55-62, 67-74, 114-121,130-140, 188-197, 208-217, 226-232, 265-287, 292-299, 301-319, 372-394,400-410, 421-427 and 12-56 of Seq ID No 49; 6-12, 44-51, 53-60, 67-88,91-100, 104-123, 137-142, 148-158, 161-168, 175-201, 204-210, 222-231,239-253, 258-264, 272-282 and 60-138 of Seq ID No 50; 4-63, 69-104,110-121, 124-131, 134-152, 161-187, 204-221, 223-237, 239-296, 298-310,331-365, 380-405, 423-451, 470-552, 554-562, 574-581, 592-649, 651-658,661-671, 673-707, 713-734, 741-748, 758-765, 773-790 and 509-528 of SeqID No 51; 89-94, 102-115, 123-129, 181-188, 200-206, 211-235, 239-249,267-281, 295-310, 316-321, 331-341, 344-359, 365-386, 409-422, 443-453,495-506, 514-521, 539-547, 553-560, 563-570, 586-596, 621-626, 633-638,651-657, 666-683, 697-705, 731-739, 761-768, 865-883 and 213-265 of SeqID No 52; 5-20, 24-34, 37-43, 92-102, 134-139, 156-162, 184-191,193-205, 207-213, 225-231, 241-247, 259-267, 269-286, 337-350, 365-372,378-386, 399-413, 415-421, 447-457, 467-481 and 145-183 of Seq ID No 53;12-19, 29-41, 43-57, 80-98, 106-141, 143-156, 172-183, 185-210, 214-220,226-234, 278-287 and 237-287 of Seq ID No 54; 5-12, 32-48, 50-72, 75-81,88-94 and 16-40 of Seq ID No 55; 4-21, 29-42, 48-62, 65-80, 95-101,103-118, 122-130, 134-140, 143-152, 155-165, 182-192, 198-208, 232-247,260-268, 318-348, 364-369, 380-391, 403-411, 413-424 and 208-230 of SeqID No 56; 4-18, 65-75, 82-92, 123-140, 144-159, 166-172, 188-194 and174-195 of Seq ID No 57; 7-20, 58-71, 94-101, 110-119, 199-209, 231-242,247-254, 267-277, 282-290, 297-306, 313-319, 333-342, 344-369, 390-402,414-431, 436-448, 462-471 and 310-350 of Seq ID No 58; 4-25, 37-44,53-59, 72-78, 86-99, 119-128, 197-203, 209-218, 220-226, 233-244,246-254, 264-271, 277-289, 407-430, 437-445, 464-472, 482-488, 503-509and 308-331 of Seq ID No 59; 4-12, 14-43, 52-58 and 43-58 of Seq ID No60; 4-14, 21-29, 35-49 and 38-50 of Seq ID No 61; 4-19, 31-37, 58-72,94-108 and 1-72 of Seq ID No 62.

The present invention also provides a process for producing a S.epidermidis hyperimmune serum reactive antigen or a fragment thereofaccording to the present invention comprising expressing one or more ofthe nucleic acid molecules according to the present invention in asuitable expression system.

Moreover, the present invention provides a process for producing a cell,which expresses a S. epidermidis hyperimmune serum reactive antigen or afragment thereof according to the present invention comprisingtransforming or transfecting a suitable host cell with the vectoraccording to the present invention.

According to the present invention a pharmaceutical composition,especially a vaccine, comprising a hyperimmune serum-reactive antigen ora fragment thereof as defined in the present invention or a nucleic acidmolecule as defined in the present invention is provided.

In a preferred embodiment the pharmaceutical composition furthercomprises an immunostimulatory substance, preferably selected from thegroup comprising polycationic polymers, especially polycationicpeptides, immunostimulatory deoxynucleotides (ODNs), peptides containingat least two LysLeuLys motifs, especially KLKLSKLK (SEQ ID NO:63),neuroactive compounds, espedully human growth hormone, alumn, Freund'scomplete or incomplete adjuvants or combinations thereof.

In a more preferred embodiment the immunostimulatory substance is acombination of either a polycationic polymer and immunostimulatorydeoxynucleotides or of a peptide containing at least two LysLeuLysmotifs and immunostimulatory deoxynucleotides.

In a still more preferred embodiment the polycationic polymer is apolycationic peptide, especially polyarginine.

According to the present invention the use of a nucleic acid moleculeaccording to the present invention or a hyperimmune serum-reactiveantigen or fragment thereof according to the present invention for themanufacture of a pharmaceutical preparation, especially for themanufacture of a vaccine against S. epidermidis infection, is provided.

Also an antibody, or at least an effective part thereof, which binds atleast to a selective part of the hyperimmune serum-reactive antigen or afragment thereof according to the present invention is providedherewith.

In a preferred embodiment the antibody is a monoclonal antibody.

In another preferred embodiment the effective part of the antibodycomprises Fab fragments.

In a further preferred embodiment the antibody is a chimeric antibody.

In a still preferred embodiment the antibody is a humanized antibody.

The present invention also provides a hybridoma cell line, whichproduces an antibody according to the present invention.

Moreover, the present invention provides a method for producing anantibody according to the present invention, characterized by thefollowing steps:

-   -   initiating an immune response in a non-human animal by        administrating an hyperimmune serum-reactive antigen or a        fragment thereof, as defined in the invention, to said animal,    -   removing an antibody containing body fluid from said animal, and    -   producing the antibody by subjecting said antibody containing        body fluid to further purification steps.

Accordingly, the present invention also provides a method for producingan antibody according to the present invention, characterized by thefollowing steps:

-   -   initiating an immune response in a non-human animal by        administrating an hyperimmune serum-reactive antigen or a        fragment thereof, as defined in the present invention, to said        animal,    -   removing the spleen or spleen cells from said animal,    -   producing hybridoma cells of said spleen or spleen cells,    -   selecting and cloning hybridoma cells specific for said        hyperimmune serum-reactive antigens or a fragment thereof,    -   producing the antibody by cultivation of said cloned hybridoma        cells and optionally further purification steps.

The antibodies provided or produced according to the above methods maybe used for the preparation of a medicament for treating or preventingS. epidermidis infections.

According to another aspect the present invention provides anantagonist, which binds to a hyperimmune serum-reactive antigen or afragment thereof according to the present invention.

Such an antagonist capable of binding to a hyperimmune serum-reactiveantigen or fragment thereof according to the present invention may beidentified by a method comprising the following steps:

-   a) contacting an isolated or immobilized hyperimmune serum-reactive    antigen or a fragment thereof according to the present invention    with a candidate antagonist under conditions to permit binding of    said candidate antagonist to said hyperimmune serum-reactive antigen    or fragment, in the presence of a component capable of providing a    detectable signal in response to the binding of the candidate    antagonist to said hyperimmune serum reactive antigen or fragment    thereof; and-   b) detecting the presence or absence of a signal generated in    response to the binding of the antagonist to the hyperimmune serum    reactive antigen or the fragment thereof.

An antagonist capable of reducing or inhibiting the interaction activityof a hyperimmune serum-reactive antigen or a fragment thereof accordingto the present invention to its interaction partner may be identified bya method comprising the following steps:

-   a) providing a hyperimmune serum reactive antigen or a hyperimmune    fragment thereof according to the present invention,-   b) providing an interaction partner to said hyperimmune serum    reactive antigen or a fragment thereof, especially an antibody    according to the present invention,-   c) allowing interaction of said hyperimmune serum reactive antigen    or fragment thereof to said interaction partner to form an    interaction complex,-   d) providing a candidate antagonist,-   e) allowing a competition reaction to occur between the candidate    antagonist and the interaction complex,-   f) determining whether the candidate antagonist inhibits or reduces    the interaction activities of the hyperimmune serum reactive antigen    or the fragment thereof with the interaction partner.

The hyperimmune serum reactive antigens or fragments thereof accordingto the present invention may be used for the isolation and/orpurification and/or identification of an interaction partner of saidhyperimmune serum reactive antigen or fragment thereof.

The present invention also provides a process for in vitro diagnosing adisease related to expression of a hyperimmune serum-reactive antigen ora fragment thereof according to the present invention comprisingdetermining the presence of a nucleic acid sequence encoding saidhyperimmune serum reactive antigen or fragment thereof according to thepresent invention or the presence of the hyperimmune serum reactiveantigen or fragment thereof according to the present invention.

The present invention also provides a process for in vitro diagnosis ofa bacterial infection, especially a S. epidermidis infection, comprisinganalyzing for the presence of a nucleic acid sequence encoding saidhyperimmune serum reactive antigen or fragment thereof according to thepresent invention or the presence of the hyperimmune serum reactiveantigen or fragment thereof according to the present invention.

Moreover, the present invention provides the use of a hyperimmune serumreactive antigen or fragment thereof according to the present inventionfor the generation of a peptide binding to said hyperimmune serumreactive antigen or fragment thereof, wherein the peptide is ananticaline.

The present invention also provides the use of a hyperimmuneserum-reactive antigen or fragment thereof according to the presentinvention for the manufacture of a functional nucleic acid, wherein thefunctional nucleic acid is selected from the group comprising aptamersand spiegelmers.

The nucleic acid molecule according to the present invention may also beused for the manufacture of a functional ribonucleic acid, wherein thefunctional ribonucleic acid is selected from the group comprisingribozymes, antisense nucleic acids and siRNA.

The present invention advantageously provides an efficient and relevantset of isolated nucleic acid molecules and their encoded hyperimmuneserum reactive antigens or fragments thereof identified from S.epidermidis using an antibody preparation from a human plasma pool andsurface expression libraries derived from the genome of S. epidermidis.Thus, the present invention fulfils a widely felt demand for S.epidermidis antigens, vaccines, diagnostics and products useful inprocedures for preparing antibodies and for identifying compoundseffective against S. epidermidis infection.

An effective vaccine should be composed of proteins or polypeptides,which are expressed by all strains and are able to induce high affinity,abundant antibodies against cell surface components of S. epidermidis.The antibodies should be IgG1 and/or IgG3 for opsonization, and any IgGsub-type and IgA for neutralisation of adherence and toxin action. Achemically defined vaccine must be definitely superior compared to awhole cell vaccine (attenuated or killed), since components of S.epidermidis, which might cross-react with human tissues or inhibitopsonization can be eliminated, and the individual proteins inducingprotective antibodies and/or a protective immune response can beselected.

The approach, which has been employed for the present invention, isbased on the interaction of staphylococcal proteins or peptides with theantibodies present in human sera. The antibodies produced against S.epidermidis by the human immune system and present in human sera areindicative of the in vivo expression of the antigenic proteins and theirimmunogenicity. In addition, the antigenic proteins as identified by thebacterial surface display expression libraries using pools ofpre-selected sera, are processed in a second and third round ofscreening by individual selected or generated sera. Thus the presentinvention supplies an efficient and relevant set of staphyloococcalantigens as a pharmaceutical composition, especially a vaccinepreventing infection by S. epidermidis.

In the antigen identification program for identifying a relevant andefficient set of antigens according to the present invention, threedifferent bacterial surface expression libraries are screened with aserum pool derived from a serum collection, which has been testedagainst antigenic compounds of S. epidermidis, such as whole cellextracts and culture supernatant proteins in order to be consideredhyperimmune and therefore relevant in the screening method applied forthe present invention. The antibodies produced against staphyloococci bythe human immune system and present in human sera are indicative of thein vivo expression of the antigenic proteins and their immunogenicity.

The expression libraries as used in the present invention should allowexpression of all potential antigens, e.g. derived from all surfaceproteins of S. epidermidis. Bacterial surface display libraries will berepresented by a recombinant library of a bacterial host displaying a(total) set of expressed peptide sequences of staphylococci on a numberof selected outer membrane proteins (LamB, FhuA) at the bacterial hostmembrane {Georgiou, G., 1997; Etz, H. et al., 2001}. One of theadvantages of using recombinant expression libraries is that theidentified hyperimmune serum-reactive antigens may be instantly producedby expression of the coding sequences of the screened and selectedclones expressing the hyperimmune serum-reactive antigens withoutfurther recombinant DNA technology or cloning steps necessary.

The comprehensive set of antigens identified by the described programaccording to the present invention is analysed further by one or moreadditional rounds of screening. Therefore individual antibodypreparations or antibodies generated against selected peptides, whichwere identified as immunogenic are used. According to a preferredembodiment the individual antibody preparations for the second round ofscreening are derived from patients who have suffered from an acuteinfection with staphylococci, especially from patients who show anantibody titer above a certain minimum level, for example an antibodytiter being higher than 80 percentile, preferably higher than 90percentile, especially higher than 95 percentile of the human (patientor healthy individual) sera tested. Using such high titer individualantibody preparations in the second screening round allows a veryselective identification of the hyperimmune serum-reactive antigens andfragments thereof from S. epidermidis.

Following the screening procedure, the selected antigenic proteins,expressed as recombinant proteins or in vitro translated products, incase it can not be expressed in prokaryotic expression systems, or theidentified antigenic peptides (produced synthetically) are tested in asecond screening by a series of ELISA and Western blotting assays forthe assessment of their immunogenicity with a large human serumcollection (>100 uninfected, >50 patients sera).

It is important that the individual antibody preparations (which mayalso be the selected serum) allow a selective identification of the mostpromising candidates of all the hyperimmune serum-reactive antigens fromall the promising candidates from the first round. Therefore, preferablyat least 10 individual antibody preparations (i.e. antibody preparations(e.g. sera) from at least 10 different individuals having suffered froman infection to the chosen pathogen) should be used in identifying theseantigens in the second screening round. Of course, it is possible to usealso less than 10 individual preparations, however, selectivity of thestep may not be optimal with a low number of individual antibodypreparations. On the other hand, if a given hyperimmune serum-reactiveantigen (or an antigenic fragment thereof) is recognized by at least 10individual antibody preparations, preferably at least 30, especially atleast 50 individual antibody preparations, identification of thehyperimmune serum-reactive antigen is also selective enough for a properidentification. Hyperimmune serum-reactivity may of course be testedwith as many individual preparations as possible (e.g. with more than100 or even with more than 1,000).

Therefore, the relevant portion of the hyperimmune serum-reactiveantibody preparations according to the method of the present inventionshould preferably be at least 10, more preferred at least 30, especiallyat least 50 individual antibody preparations. Alternatively (or incombination) hyperimmune serum-reactive antigens may preferably be alsoidentified with at least 20%, preferably at least 30%, especially atleast 40% of all individual antibody preparations used in the secondscreening round.

According to a preferred embodiment of the present invention, the serafrom which the individual antibody preparations for the second round ofscreening are prepared (or which are used as antibody preparations), areselected by their titer against S. epidermidis (e.g. against apreparation of this pathogen, such as a lysate, cell wall components andrecombinant proteins). Preferably, some are selected with a total IgAtiter above 4,000 U, especially above 6,000 U, and/or an IgG titer above10,000 U, especially above 12,000 U (U=units, calculated from the OD405nm reading at a given dilution) when the whole organism (total lysate orwhole cells) is used as antigen in the ELISA.

The antibodies produced against staphylococci by the human immune systemand present in human sera are indicative of the in vivo expression ofthe antigenic proteins and their immunogenicity. The recognition oflinear epitopes by antibodies can be based on sequences as short as 4-5amino acids. Of course it does not necessarily mean that these shortpeptides are capable of inducing the given antibody in vivo. For thatreason the defined epitopes, polypeptides and proteins are further to betested in animals (mainly in mice) for their capacity to induceantibodies against the selected proteins in vivo.

The preferred antigens are located on the cell surface or are secreted,and are therefore accessible extracellularly. Antibodies against cellwall proteins are expected to serve two purposes: to inhibit adhesionand to promote phagocytosis. Antibodies against secreted proteins arebeneficial in neutralisation of their function as toxin or virulencecomponent. It is also known that bacteria communicate with each otherthrough secreted proteins. Neutralizing antibodies against theseproteins will interrupt growth-promoting cross-talk between or withinstreptococcal species. Bioinformatic analyses (signal sequences, cellwall localisation signals, transmembrane domains) proved to be veryuseful in assessing cell surface localisation or secretion. Theexperimental approach includes the isolation of antibodies with thecorresponding epitopes and proteins from human serum, and the generationof immune sera in mice against (poly)peptides selected by the bacterialsurface display screens. These sera are then used in a third round ofscreening as reagents in the following assays: cell surface staining ofstaphylococci grown under different conditions (FACS, microscopy),determination of neutralizing capacity (toxin, adherence), and promotionof opsonization and phagocytosis (in vitro phagocytosis assay).

For that purpose, bacterial E. coli clones are directly injected intomice and immune sera are taken and tested in the relevant in vitro assayfor functional opsonic or neutralizing antibodies. Alternatively,specific antibodies may be purified from human or mouse sera usingpeptides or proteins as substrate.

Host defence against S. epidermidis relies mainly on innateimmunological mechanisms. Inducing high affinity antibodies of theopsonic and neutralizing type by vaccination helps the innate immunesystem to eliminate bacteria and toxins. This makes the method accordingto the present invention an optimal tool for the identification ofstaphylococcal antigenic proteins.

The skin and mucous membranes are formidable barriers against invasionby staphylococci. However, once the skin or the mucous membranes arebreached the first line of non-adaptive cellular defence begins itsco-ordinate action through complement and phagocytes, especially thepolymorphonuclear leukocytes (PMNs). These cells can be regarded as thecornerstones in eliminating invading bacteria. As staphylococci areprimarily extracellular pathogens, the major anti-staphylococcaladaptive response comes from the humoral arm of the immune system, andis mediated through three major mechanisms: promotion of opsonization,toxin neutralisation, and inhibition of adherence. It is believed thatopsonization is especially important, because of its requirement for aneffective phagocytosis. For efficient opsonization the microbial surfacehas to be coated with antibodies and complement factors for recognitionby PMNs through receptors to the Fc fragment of the IgG molecule or toactivated C3b. After opsonization, staphyloococci are phagocytosed andkilled. Antibodies bound to specific antigens on the cell surface ofbacteria serve as ligands for the attachment to PMNs and to promotephagocytosis. The very same antibodies bound to the adhesins and othercell surface proteins are expected to neutralize adhesion and preventcolonization. The selection of antigens as provided by the presentinvention is thus well suited to identify those that will lead toprotection against infection in an animal model or in humans.

According to the antigen identification method used herein, the presentinvention can surprisingly provide a set of novel nucleic acids andnovel hyperimmune serum reactive antigens and fragments thereof of S.epidermidis, among other things, as described below. According to oneaspect, the invention particularly relates to the nucleotide sequencesencoding hyperimmune serum reactive antigens which sequences are setforth in the Sequence listing Seq ID No: 1-31 and the correspondingencoded amino acid sequences representing hyperimmune serum reactiveantigens are set forth in the Sequence Listing Seq ID No 32-62.

In a preferred embodiment of the present invention, a nucleic acidmolecule is provided which exhibits 70% identity over their entirelength to a nucleotide sequence set forth with Seq ID No 1, 4, 6-9,11-13, 15, 17, 19, 21, 25-26, 28-31. Most highly preferred are nucleicacids that comprise a region that is at least 80% or at least 85%identical over their entire length to a nucleic acid molecule set forthwith Seq ID No 1, 4, 6-9, 11-13, 15, 17, 19, 21, 25-26, 28-31. In thisregard, nucleic acid molecules at least 90%, 91%, 92%, 93%, 94%, 95%, or96% identical over their entire length to the same are particularlypreferred. Furthermore, those with at least 97% are highly preferred,those with at least 98% and at least 99% are particularly highlypreferred, with at least 99% or 99.5% being the more preferred, with100% identity being especially preferred. Moreover, preferredembodiments in this respect are nucleic acids which encode hyperimmuneserum reactive antigens or fragments thereof (polypeptides) which retainsubstantially the same biological function or activity as the maturepolypeptide encoded by said nucleic acids set forth in the Seq ID No 1,4, 6-9, 11-13, 15, 17, 19, 21, 25-26, 28-31.

Identity, as known in the art and used herein, is the relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. Identity canbe readily calculated. While there exist a number of methods to measureidentity between two polynucleotide or two polypeptide sequences, theterm is well known to skilled artisans (e.g. Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987). Preferredmethods to determine identity are designed to give the largest matchbetween the sequences tested. Methods to determine identity are codifiedin computer programs. Preferred computer program methods to determineidentity between two sequences include, but are not limited to, GCGprogram package {Devereux, J. et al., 1984}, BLASTP, BLASTN, and FASTA{Altschul, S. et al., 1990}.

According to another aspect of the invention, nucleic acid molecules areprovided which exhibit at least 96% identity to the nucleic acidsequence set forth with Seq ID No 2-3, 5, 10, 14, 16, 18, 22-24, 27.

According to a further aspect of the present invention, nucleic acidmolecules are provided which are identical to the nucleic acid sequencesset forth with Seq ID No 20.

The nucleic acid molecules according to the present invention can as asecond alternative also be a nucleic acid molecule which is at leastessentially complementary to the nucleic acid described as the firstalternative above. As used herein complementary means that a nucleicacid strand is base pairing via Watson-Crick base pairing with a secondnucleic acid strand. Essentially complementary as used herein means thatthe base pairing is not occurring for all of the bases of the respectivestrands but leaves a certain number or percentage of the bases unpairedor wrongly paired. The percentage of correctly pairing bases ispreferably at least 70%, more preferably 80%, even more preferably 90%and most preferably any percentage higher than 90%. It is to be notedthat a percentage of 70% matching bases is considered as homology andthe hybridization having this extent of matching base pairs isconsidered as stringent. Hybridization conditions for this kind ofstringent hybridization may be taken from Current Protocols in MolecularBiology (John Wiley and Sons, Inc., 1987). More particularly, thehybridization conditions can be as follows:

-   -   Hybridization performed e.g. in 5×SSPE, 5×Denhardt's reagent,        0.1% SDS, 100 g/mL sheared DNA at 68° C.    -   Moderate stringency wash in 0.2×SSC, 0.1% SDS at 42° C.    -   High stringency wash in 0.1×SSC, 0.1% SDS at 68° C.

Genomic DNA with a GC content of 50% has an approximate TM of 96° C. For1% mismatch, the TM is reduced by approximately 1° C.

In addition, any of the further hybridization conditions describedherein are in principle applicable as well.

Of course, all nucleic acid sequence molecules which encode the samepolypeptide molecule as those identified by the present invention areencompassed by any disclosure of a given coding sequence, since thedegeneracy of the genetic code is directly applicable to unambiguouslydetermine all possible nucleic acid molecules which encode a givenpolypeptide molecule, even if the number of such degenerated nucleicacid molecules may be high. This is also applicable for fragments of agiven polypeptide, as long as the fragments encode a polypeptide beingsuitable to be used in a vaccination connection, e.g. as an active orpassive vaccine.

The nucleic acid molecule according to the present invention can as athird alternative also be a nucleic acid which comprises a stretch of atleast 15 bases of the nucleic acid molecule according to the first andsecond alternative of the nucleic acid molecules according to thepresent invention as outlined above. Preferably, the bases form acontiguous stretch of bases. However, it is also within the scope of thepresent invention that the stretch consists of two or more moieties,which are separated by a number of bases.

The present nucleic acids may preferably consist of at least 20, evenmore preferred at least 30, especially at least 50 contiguous bases fromthe sequences disclosed herein. The suitable length may easily beoptimized due to the planned area of use (e.g. as (PCR) primers, probes,capture molecules (e.g. on a (DNA) chip), etc.). Preferred nucleic acidmolecules contain at least a contiguous 15 base portion of one or moreof the predicted immunogenic amino acid sequences listed in Table 1,especially the sequences of Table 1 with scores of more than 10,preferably more than 20, especially with a score of more than 25.Specifically preferred are nucleic acids containing a contiguous portionof a DNA sequence of any sequence in the sequence protocol of thepresent application which shows 1 or more, preferably more than 2,especially more than 5, non-identical nucleic acid residues compared tothe published Staphylococcus epidermidis strain RP62A genome(http://www.tigr.org/tdb/mdb/mdbinprogress.html) and/or any otherpublished S. epidermidis genome sequence or parts thereof. Specificallypreferred non-identical nucleic acid residues are residues, which leadto a non-identical amino acid residue. Preferably, the nucleic acidsequences encode for polypeptides having at least 1, preferably at least2, preferably at least three different amino acid residues compared tothe published S. epidermidis counterparts mentioned above. Also suchisolated polypeptides, being fragments of the proteins (or the wholeprotein) mentioned herein e.g. in the sequence listing, having at least6, 7, or 8 amino acid residues and being encoded by these nucleic acidsare preferred.

The nucleic acid molecule according to the present invention can as afourth alternative also be a nucleic acid molecule which anneals understringent hybridisation conditions to any of the nucleic acids of thepresent invention according to the above outlined first, second, andthird alternative. Stringent hybridisation conditions are typicallythose described herein.

Finally, the nucleic acid molecule according to the present inventioncan as a fifth alternative also be a nucleic acid molecule which, butfor the degeneracy of the genetic code, would hybridise to any of thenucleic acid molecules according to any nucleic acid molecule of thepresent invention according to the first, second, third, and fourthalternative as outlined above. This kind of nucleic acid molecule refersto the fact that preferably the nucleic acids according to the presentinvention code for the hyperimmune serum reactive antigens or fragmentsthereof according to the present invention. This kind of nucleic acidmolecule is particularly useful in the detection of a nucleic acidmolecule according to the present invention and thus the diagnosis ofthe respective microorganisms such as S. epidermidis and any disease ordiseased condition where this kind of microorganims is involved.Preferably, the hybridisation would occur or be preformed understringent conditions as described in connection with the fourthalternative described above.

Nucleic acid molecule as used herein generally refers to any ribonucleicacid molecule or deoxyribonucleic acid molecule, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, nucleic acidmolecule as used herein refers to, among other, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded RNA, and RNA that is a mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded, or a mixture of single- and double-stranded regions. Inaddition, nucleic acid molecule as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide. Asused herein, the term nucleic acid molecule includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“nucleic acid molecule” as that term is intended herein. Moreover, DNAsor RNAs comprising unusual bases, such as inosine, or modified bases,such as tritylated bases, to name just two examples, are nucleic acidmolecule as the term is used herein. It will be appreciated that a greatvariety of modifications have been made to DNA and RNA that serve manyuseful purposes known to those of skill in the art. The term nucleicacid molecule as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of nucleic acid molecule,as well as the chemical forms of DNA and RNA characteristic of virusesand cells, including simple and complex cells, inter alia. The termnucleic acid molecule also embraces short nucleic acid molecules oftenreferred to as oligonucleotide(s). “Polynucleotide” and “nucleic acid”or “nucleic acid molecule” are often used interchangeably herein.

Nucleic acid molecules provided in the present invention also encompassnumerous unique fragments, both longer and shorter than the nucleic acidmolecule sequences set forth in the sequencing listing of the S.epidermidis coding regions, which can be generated by standard cloningmethods. To be unique, a fragment must be of sufficient size todistinguish it from other known nucleic acid sequences, most readilydetermined by comparing any selected S. epidermidis fragment to thenucleotide sequences in computer databases such as GenBank.

Additionally, modifications can be made to the nucleic acid moleculesand polypeptides that are encompassed by the present invention. Forexample, nucleotide substitutions can be made which do not affect thepolypeptide encoded by the nucleic acid, and thus any nucleic acidmolecule which encodes a hyperimmune serum reactive antigen or fragmentsthereof is encompassed by the present invention.

Furthermore, any of the nucleic acid molecules encoding hyperimmuneserum reactive antigens or fragments thereof provided by the presentinvention can be functionally linked, using standard techniques such asstandard cloning techniques, to any desired regulatory sequences,whether a S. epidermidis regulatory sequence or a heterologousregulatory sequence, heterologous leader sequence, heterologous markersequence or a heterologous coding sequence to create a fusion protein.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA or cRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or produced bychemical synthetic techniques or by a combination thereof. The DNA maybe triple-stranded, double-stranded or single-stranded. Single-strandedDNA may be the coding strand, also known as the sense strand, or it maybe the non-coding strand, also referred to as the anti-sense strand.

The present invention further relates to variants of the herein abovedescribed nucleic acid molecules which encode fragments, analogs andderivatives of the hyperimmune serum reactive antigens and fragmentsthereof having a deducted S. epidermidis amino acid sequence set forthin the Sequence Listing. A variant of the nucleic acid molecule may be anaturally occurring variant such as a naturally occurring allelicvariant, or it may be a variant that is not known to occur naturally.Such non-naturally occurring variants of the nucleic acid molecule maybe made by mutagenesis techniques, including those applied to nucleicacid molecules, cells or organisms.

Among variants in this regard are variants that differ from theaforementioned nucleic acid molecules by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or additions. Preferred are nucleic acid molecules encoding avariant, analog, derivative or fragment, or a variant, analogue orderivative of a fragment, which have a S. epidermidis sequence as setforth in the Sequence Listing, in which several, a few, 5 to 10, 1 to 5,1 to 3, 2, 1 or no amino acid(s) is substituted, deleted or added, inany combination. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the S. epidermidis polypeptides set forthin the Sequence Listing. Also especially preferred in this regard areconservative substitutions.

The peptides and fragments according to the present invention alsoinclude modified epitopes wherein preferably one or two of the aminoacids of a given epitope are modified or replaced according to the rulesdisclosed in e.g. {Tourdot, S. et al., 2000}, as well as the nucleicacid sequences encoding such modified epitopes.

It is clear that also epitopes derived from the present epitopes byamino acid exchanges improving, conserving or at least not significantlyimpeding the T cell activating capability of the epitopes are covered bythe epitopes according to the present invention. Therefore the presentepitopes also cover epitopes, which do not contain the original sequenceas derived from S. epidermidis, but trigger the same or preferably animproved T cell response. These epitope are referred to as“heteroclitic”; they need to have a similar or preferably greateraffinity to MHC/HLA molecules, and the need the ability to stimulate theT cell receptors (TCR) directed to the original epitope in a similar orpreferably stronger manner.

Heteroclitic epitopes can be obtained by rational design i.e. takinginto account the contribution of individual residues to binding toMHC/HLA as for instance described by {Rammensee, H. et al., 1999},combined with a systematic exchange of residues potentially interactingwith the TCR and testing the resulting sequences with T cells directedagainst the original epitope. Such a design is possible for a skilledman in the art without much experimentation.

Another possibility includes the screening of peptide libraries with Tcells directed against the original epitope. A preferred way is thepositional scanning of synthetic peptide libraries. Such approaches havebeen described in detail for instance by {Hemmer, B. et al., 1999} andthe references given therein.

As an alternative to epitopes represented by the present derived aminoacid sequences or heteroclitic epitopes, also substances mimicking theseepitopes e.g. “peptidemimetica” or “retroinverso-peptides” can beapplied.

Another aspect of the design of improved epitopes is their formulationor modification with substances increasing their capacity to stimulate Tcells. These include T helper cell epitopes, lipids or liposomes orpreferred modifications as described in WO 01/78767.

Another way to increase the T cell stimulating capacity of epitopes istheir formulation with immune stimulating substances for instancecytokines or chemokines like interleukin-2, -7, -12, -18, class I and IIinterferons (IFN), especially IFN-gamma, GM-CSF, TNF-alpha, flt3-ligandand others.

As discussed additionally herein regarding nucleic acid molecule assaysof the invention, for instance, nucleic acid molecules of the inventionas discussed above, may be used as a hybridization probe for RNA, cDNAand genomic DNA to isolate full-length cDNAs and genomic clones encodingpolypeptides of the present invention and to isolate cDNA and genomicclones of other genes that have a high sequence similarity to thenucleic acid molecules of the present invention. Such probes generallywill comprise at least 15 bases. Preferably, such probes will have atleast 20, at least 25 or at least 30 bases, and may have at least 50bases. Particularly preferred probes will have at least 30 bases, andwill have 50 bases or less, such as 30, 35, 40, 45, or 50 bases.

For example, the coding region of a nucleic acid molecule of the presentinvention may be isolated by screening a relevant library using theknown DNA sequence to synthesize an oligonucleotide probe. A labeledoligonucleotide having a sequence complementary to that of a gene of thepresent invention is then used to screen a library of cDNA, genomic DNAor mRNA to determine to which members of the library the probehybridizes.

The nucleic acid molecules and polypeptides of the present invention maybe employed as reagents and materials for development of treatments ofand diagnostics for disease, particularly human disease, as furtherdiscussed herein relating to nucleic acid molecule assays, inter alia.

The nucleic acid molecules of the present invention that areoligonucleotides can be used in the processes herein as described, butpreferably for PCR, to determine whether or not the S. epidermidis genesidentified herein in whole or in part are present and/or transcribed ininfected tissue such as blood. It is recognized that such sequences willalso have utility in diagnosis of the stage of infection and type ofinfection the pathogen has attained. For this and other purposes thearrays comprising at least one of the nucleic acids according to thepresent invention as described herein, may be used.

The nucleic acid molecules according to the present invention may beused for the detection of nucleic acid molecules and organisms orsamples containing these nucleic acids. Preferably such detection is fordiagnosis, more preferable for the diagnosis of a disease related orlinked to the present or abundance of S. epidermidis.

Eukaryotes (herein also “individual(s)”), particularly mammals, andespecially humans, infected with S. epidermidis may be identifiable bydetecting any of the nucleic acid molecules according to the presentinvention detected at the DNA level by a variety of techniques.Preferred nucleic acid molecules candidates for distinguishing a S.epidermidis from other organisms can be obtained.

The different polypeptides described herein can have therapeutic and/ordiagnostic utilities. The present application identifies differentimmunogenic polypeptides, and immunogenic polypeptide regions,characteristic of S. epi. An immunogenic polypeptide region can bepresent by itself or part of a longer length polypeptide. Thepolypeptides and polypeptide regions can be used in diagnosticapplications to provide an indication as to whether a person is, or hasbeen, infected with S. epi. For example, a polypeptide containing an S.epi immunogenic region can be used to generate S. epi antibodies, whichcan be used to detect the presence of S. epi in serum; and a polypeptidecontaining an S. epi immunogenic region can be used to detect thepresence of S. epi. antibodies in serum.

The invention provides a process for diagnosing disease, arising frominfection with S. epidermidis, comprising determining from a sampleisolated or derived from an individual an increased level of expressionof a nucleic acid molecule having the sequence of a nucleic acidmolecule set forth in the Sequence Listing. Expression of nucleic acidmolecules can be measured using any one of the methods well known in theart for the quantitation of nucleic acid molecules, such as, forexample, PCR, RT-PCR, Rnase protection, Northern blotting, otherhybridisation methods and the arrays described herein.

Isolated as used herein means separated “by the hand of man” from itsnatural state; i.e., that, if it occurs in nature, it has been changedor removed from its original environment, or both. For example, anaturally occurring nucleic acid molecule or a polypeptide naturallypresent in a living organism in its natural state is not “isolated,” butthe same nucleic acid molecule or polypeptide separated from thecoexisting materials of its natural state is “isolated”, as the term isemployed herein. As part of or following isolation, such nucleic acidmolecules can be joined to other nucleic acid molecules, such as DNAs,for mutagenesis, to form fusion proteins, and for propagation orexpression in a host, for instance. The isolated nucleic acid molecules,alone or joined to other nucleic acid molecules such as vectors, can beintroduced into host cells, in culture or in whole organisms. Introducedinto host cells in culture or in whole organisms, such DNAs still wouldbe isolated, as the term is used herein, because they would not be intheir naturally occurring form or environment. Similarly, the nucleicacid molecules and polypeptides may occur in a composition, such as amedia formulations, solutions for introduction of nucleic acid moleculesor polypeptides, for example, into cells, compositions or solutions forchemical or enzymatic reactions, for instance, which are not naturallyoccurring compositions, and, therein remain isolated nucleic acidmolecules or polypeptides within the meaning of that term as it isemployed herein.

The nucleic acids according to the present invention may be chemicallysynthesized. Alternatively, the nucleic acids can be isolated from S.epidermidis by methods known to the one skilled in the art.

According to another aspect of the present invention, a comprehensiveset of novel hyperimmune serum reactive antigens and fragments thereofare provided by using the herein described antigen identificationmethod. In a preferred embodiment of the invention, a hyperimmuneserum-reactive antigen comprising an amino acid sequence being encodedby any one of the nucleic acids molecules herein described and fragmentsthereof are provided. In another preferred embodiment of the invention anovel set of hyperimmune serum-reactive antigens which comprises aminoacid sequences selected from a group consisting of the polypeptidesequences as represented in Seq ID No 32, 35, 37-40, 42-44, 46, 48, 50,52, 56-57, 59-62 and fragments thereof are provided. In a furtherpreferred embodiment of the invention hyperimmune serum-reactiveantigens, which comprise amino acid sequences selected from a groupconsisting of the polypeptide sequences as represented in Seq ID No33-34, 36, 41, 45, 47, 49, 53-55, 58 and fragments thereof are provided.In a still preferred embodiment of the invention hyperimmuneserum-reactive antigens which comprise amino acid sequences selectedfrom a group consisting of the polypeptide sequences as represented inSeq ID No 51 and fragments thereof are provided.

The hyperimmune serum reactive antigens and fragments thereof asprovided in the invention include any polypeptide set forth in theSequence Listing as well as polypeptides which have at least 70%identity to a polypeptide set forth in the Sequence Listing, preferablyat least 80% or 85% identity to a polypeptide set forth in the SequenceListing, and more preferably at least 90% similarity (more preferably atleast 90% identity) to a polypeptide set forth in the Sequence Listingand still more preferably at least 95%, 96%, 97%, 98%, 99% or 99.5%similarity (still more preferably at least 95%, 96%, 97%, 98%, 99%, or99.5% identity) to a polypeptide set forth in the Sequence Listing andalso include portions of such polypeptides with such portion of thepolypeptide generally containing at least 4 amino acids and morepreferably at least 8, still more preferably at least 30, still morepreferably at least 50 amino acids, such as 4, 8, 10, 20, 30, 35, 40, 45or 50 amino acids.

The invention also relates to fragments, analogs, and derivatives ofthese hyperimmune serum reactive antigens and fragments thereof. Theterms “fragment”, “derivative” and “analog” when referring to an antigenwhose amino acid sequence is set forth in the Sequence Listing, means apolypeptide which retains essentially the same or a similar biologicalfunction or activity as such hyperimmune serum reactive antigen andfragment thereof.

The fragment, derivative or analog of a hyperimmune serum reactiveantigen and fragment thereof may be 1) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or 2) one in which one or more of the amino acid residuesincludes a substituent group, or 3) one in which the mature hyperimmuneserum reactive antigen or fragment thereof is fused with anothercompound, such as a compound to increase the half-life of thehyperimmune serum reactive antigen and fragment thereof (for example,polyethylene glycol), or 4) one in which the additional amino acids arefused to the mature hyperimmune serum reactive antigen or fragmentthereof, such as a leader or secretory sequence or a sequence which isemployed for purification of the mature hyperimmune serum reactiveantigen or fragment thereof or a proprotein sequence. Such fragments,derivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

Among the particularly preferred embodiments of the invention in thisregard are the hyperimmune serum reactive antigens set forth in theSequence Listing, variants, analogs, derivatives and fragments thereof,and variants, analogs and derivatives of fragments. Additionally, fusionpolypeptides comprising such hyperimmune serum reactive antigens,variants, analogs, derivatives and fragments thereof, and variants,analogs and derivatives of the fragments are also encompassed by thepresent invention. Such fusion polypeptides and proteins, as well asnucleic acid molecules encoding them, can readily be made using standardtechniques, including standard recombinant techniques for producing andexpression of a recombinant polynucleic acid encoding a fusion protein.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe and Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of any polypeptide set forthin the Sequence Listing, in which several, a few, 5 to 10, 1 to 5, 1 to3, 2, 1 or no amino acid residues are substituted, deleted or added, inany combination. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the polypeptide of the present invention.Also especially preferred in this regard are conservative substitutions.Most highly preferred are polypeptides having an amino acid sequence setforth in the Sequence Listing without substitutions.

The hyperimmune serum reactive antigens and fragments thereof of thepresent invention are preferably provided in an isolated form, andpreferably are purified to homogeneity.

Also among preferred embodiments of the present invention arepolypeptides comprising fragments of the polypeptides having the aminoacid sequence set forth in the Sequence Listing, and fragments ofvariants and derivatives of the polypeptides set forth in the SequenceListing.

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the afore mentioned hyperimmune serum reactive antigen and fragmentthereof, and variants or derivative, analogs, fragments thereof. Suchfragments may be “free-standing”, i.e., not part of or fused to otheramino acids or polypeptides, or they may be comprised within a largerpolypeptide of which they form a part or region. Also preferred in thisaspect of the invention are fragments characterised by structural orfunctional attributes of the polypeptide of the present invention, i.e.fragments that comprise alpha-helix and alpha-helix forming regions,beta-sheet and beta-sheet forming regions, turn and turn-formingregions, coil and coil-forming regions, hydrophilic regions, hydrophobicregions, alpha amphipathic regions, beta-amphipathic regions, flexibleregions, surface-forming regions, substrate binding regions, and highantigenic index regions of the polypeptide of the present invention, andcombinations of such fragments. Preferred regions are those that mediateactivities of the hyperimmune serum reactive antigens and fragmentsthereof of the present invention. Most highly preferred in this regardare fragments that have a chemical, biological or other activity of thehyperimmune serum reactive antigen and fragments thereof of the presentinvention, including those with a similar activity or an improvedactivity, or with a decreased undesirable activity. Particularlypreferred are fragments comprising receptors or domains of enzymes thatconfer a function essential for viability of S. epidermidis or theability to cause disease in humans. Further preferred polypeptidefragments are those that comprise or contain antigenic or immunogenicdeterminants in an animal, especially in a human.

An antigenic fragment is defined as a fragment of the identifiedantigen, which is for itself antigenic or may be made antigenic whenprovided as a hapten. Therefore, also antigens or antigenic fragmentsshowing one or (for longer fragments) only a few amino acid exchangesare enabled with the present invention, provided that the antigeniccapacities of such fragments with amino acid exchanges are not severelydeteriorated on the exchange(s), i.e., suited for eliciting anappropriate immune response in an individual vaccinated with thisantigen and identified by individual antibody preparations fromindividual sera.

Preferred examples of such fragments of a hyperimmune serum-reactiveantigen are selected from the group consisting of peptides comprisingamino acid sequences of column “predicted immunogenic aa”, and “Locationof identified immunogenic region” of Table 1; the serum reactiveepitopes of Table 1, especially peptides comprising amino acid 6-28,54-59, 135-147, 193-205, 274-279, 284-291, 298-308, 342-347, 360-366,380-386, 408-425, 437-446, 457-464, 467-477, 504-510, 517-530, 535-543,547-553, 562-569, 573-579, 592-600, 602-613, 626-631, 638-668 and396-449 of Seq ID No 32; 5-24, 101-108, 111-117, 128-142, 170-184,205-211, 252-267, 308-316, 329-337, 345-353, 360-371, 375-389, 393-399,413-419, 429-439, 446-456, 471-485, 495-507, 541-556, 582-588, 592-602,607-617, 622-628, 630-640 and 8-21 of Seq ID No 33; 10-20, 23-33, 40-45,59-65, 72-107, 113-119, 127-136, 151-161 and 33-59 of Seq ID No 34;4-16, 28-34, 39-61, 66-79, 100-113, 120-127, 130-137, 142-148, 150-157,192-201, 203-210, 228-239, 245-250, 256-266, 268-278, 288-294, 312-322,336-344, 346-358, 388-396, 399-413, 425-430, 445-461, 464-470, 476-482,486-492, 503-511, 520-527, 531-541, 551-558, 566-572, 609-625, 635-642,650-656, 683-689, 691-705, 734-741, 750-767, 782-789, 802-808, 812-818,837-844, 878-885, 907-917, 930-936 and 913-933 of Seq ID No 35; 5-12,20-27, 46-78, 85-92, 104-112, 121-132, 150-167, 179-185, 200-213,221-227, 240-264, 271-279, 282-290, 311-317 and 177-206 of Seq ID No 36;18-24, 31-40, 45-51, 89-97, 100-123, 127-132, 139-153, 164-170, 184-194,200-205, 215-238, 244-255, 257-270, 272-280, 289-302, 312-318, 338-348,356-367 and 132-152 of Seq ID No 37; 7-16, 39-45, 73-83, 90-98, 118-124,130-136, 194-204, 269-280, 320-327, 373-381, 389-397, 403-408, 424-430,436-441, 463-476, 487-499, 507-514, 527-534, 540-550, 571-577, 593-599,620-629, 641-647, 650-664, 697-703, 708-717, 729-742, 773-790, 794-805,821-828, 830-837, 839-851, 858-908, 910-917, 938-947, 965-980,1025-1033, 1050-1056, 1073-1081, 1084-1098, 1106-1120, 1132-1140,1164-1170, 1185-1194, 1201-1208, 1215-1224, 1226-1234, 1267-1279,1325-1331, 1356-1364, 1394-1411, 1426-1439, 1445-1461, 1498-1504,1556-1561, 1564-1573, 1613-1639, 1648-1655, 1694-1714, 1748-1755,1778-1785, 1808-1813, 1821-1827, 1829-1837, 1846-1852, 1859-1865,1874-1883, 1895-1900, 1908-1913, 1931-1937, 1964-1981, 1995-2005,2020-2033, 2040-2047, 2103-2109, 2118-2127, 2138-2144, 2166-2175,2180-2187, 2220-2225, 2237-2242, 2247-2253, 2273-2281, 2286-2306,2314-2320, 2323-2345, 2350-2355, 2371-2384, 2415-2424, 2426-2431,2452-2472, 2584-2589, 2610-2621, 2638-2655, 2664-2670, 2681-2690,2692-2714, 2724-2730 and 687-730 of Seq ID No 38; 10-40, 53-59, 79-85,98-104, 117-122, 130-136, 144-158, 169-175, 180-185, 203-223, 232-237,243-254, 295-301 and 254-292 of Seq ID No 39; 28-50, 67-85, 93-115,120-134, 144-179, 240-249, 328-340, 354-360, 368-400, 402-417, 419-427,429-445, 447-455, 463-468, 472-480, 485-500, 502-510, 512-534, 537-546,553-558, 582-594, 619-637, 645-654, 690-709, 735-745, 749-756, 786-792,275-316 and 378-401 of Seq ID No 40; 5-16, 21-30, 33-40, 52-74, 101-108,116-122, 164-182, 185-219, 256-261, 273-279, 285-291, 297-304, 312-328,331-338, 355-362, 364-371, 373-401, 411-423 and 191-208 of Seq ID No 41;34-55, 67-74, 85-93, 105-115, 138-152, 161-171, 182-189, 197-205,213-219, 232-239, 241-248, 250-263, 272-277, 288-299 and 216-231 of SeqID No 42; 21-27, 32-37, 43-51, 67-74, 82-92, 94-100, 106-112, 140-149,153-159, 164-182, 193-215, 222-227, 260-267, 308-322, 330-340, 378-387,396-403, 417-432, 435-441, 448-465, 476-482, 488-498, 500-510 and214-280 of Seq ID No 43; 4-21, 29-52, 80-87, 104-123, 126-133, 141-157,182-189, 194-202, 214-220, 227-235, 242-252 and 33-108 of Seq ID No 44;12-18, 20-27, 29-59, 64-72, 84-90, 96-103, 109-121, 125-155, 164-177,179-186, 188-201, 216-227, 235-253, 259-274, 276-294, 296-310, 322-339,341-348, 369-379, 398-403, 409-421 and 76-96 of Seq ID No 45; 4-15,24-41, 71-80, 104-111, 113-119, 123-130, 139-149, 168-178, 187-200 and4-45 of Seq ID No 46; 13-19, 32-37, 44-56 and 1-14 of Seq ID No 47;6-11, 16-35, 75-81, 95-100, 126-139, 206-214, 225-233, 241-259, 268-276,319-325, 339-360, 371-401, 435-441, 452-459, 462-472, 491-503, 505-516,549-556, 567-580, 590-595, 612-622, 624-630, 642-648, 656-662, 687-693,698-704, 706-712, 736-750, 768-777, 784-789, 812-818, 847-858, 894-900,922-931, 938-949, 967-984, 986-992, 1027-1032, 1041-1054, 1082-1088,1091-1097, 1119-1124, 1234-1240, 1250-1258, 1274-1289, 1299-1305,1392-1398, 1400-1405, 1429-1442, 1460-1474, 1505-1514, 1531-1537,1540-1552, 1558-1571, 1582-1587, 1616-1623, 1659-1666, 1671-1677,1680-1686, 1698-1704, 1706-1712, 1768-1774, 1783-1797, 1814-1819,1849-1855, 1870-1876, 1890-1897, 1947-1953, 1972-1980, 1999-2013,2044-2051, 2068-2084, 2093-2099, 2122-2131, 2142-2147, 2156-2163,2170-2179, 2214-2220, 2235-2245, 2271-2281, 2287-2293, 2308-2317,2352-2362, 2373-2378, 2387-2407, 2442-2448, 2458-2474, 2507-2516,2531-2537, 2540-2551, 2555-2561, 2586-2599, 2617-2627, 2644-2649,2661-2675, 2685-2692, 2695-2707, 2733-2739, 2741-2747, 2774-2783,2788-2795, 2860-2870, 2891-2903, 2938-2947, 2973-2980, 2993-2999,3004-3030, 3046-3059, 3066-3077, 3082-3088, 3120-3132, 3144-3149,3153-3169, 3200-3212, 3232-3256, 3276-3290, 3308-3322, 3330-3338,3353-3360, 3363-3371, 3390-3408, 3431-3447, 3454-3484, 3503-3515,3524-3541, 3543-3550, 3560-3567, 3586-3599, 3616-3621, 3642-3647,3663-3679, 213-276, 579-621 and 1516-1559 of Seq ID No 48; 19-41, 43-49,55-62, 67-74, 114-121, 130-140, 188-197, 208-217, 226-232, 265-287,292-299, 301-319, 372-394, 400-410, 421-427 and 12-56 of Seq ID No 49;6-12, 44-51, 53-60, 67-88, 91-100, 104-123, 137-142, 148-158, 161-168,175-201, 204-210, 222-231, 239-253, 258-264, 272-282 and 60-138 of SeqID No 50; 4-63, 69-104, 110-121, 124-131, 134-152, 161-187, 204-221,223-237, 239-296, 298-310, 331-365, 380-405, 423-451, 470-552, 554-562,574-581, 592-649, 651-658, 661-671, 673-707, 713-734, 741-748, 758-765,773-790 and 509-528 of Seq ID No 51; 89-94, 102-115, 123-129, 181-188,200-206, 211-235, 239-249, 267-281, 295-310, 316-321, 331-341, 344-359,365-386, 409-422, 443-453, 495-506, 514-521, 539-547, 553-560, 563-570,586-596, 621-626, 633-638, 651-657, 666-683, 697-705, 731-739, 761-768,865-883 and 213-265 of Seq ID No 52; 5-20, 24-34, 37-43, 92-102,134-139, 156-162, 184-191, 193-205, 207-213, 225-231, 241-247, 259-267,269-286, 337-350, 365-372, 378-386, 399-413, 415-421, 447-457, 467-481and 145-183 of Seq ID No 53; 12-19, 29-41, 43-57, 80-98, 106-141,143-156, 172-183, 185-210, 214-220, 226-234, 278-287 and 237-287 of SeqID No 54; 5-12, 32-48, 50-72, 75-81, 88-94 and 16-40 of Seq ID No 55;4-21, 29-42, 48-62, 65-80, 95-101, 103-118, 122-130, 134-140, 143-152,155-165, 182-192, 198-208, 232-247, 260-268, 318-348, 364-369, 380-391,403-411, 413-424 and 208-230 of Seq ID No 56; 4-18, 65-75, 82-92,123-140, 144-159, 166-172, 188-194 and 174-195 of Seq ID No 57; 7-20,58-71, 94-101, 110-119, 199-209, 231-242, 247-254, 267-277, 282-290,297-306, 313-319, 333-342, 344-369, 390-402, 414-431, 436-448, 462-471and 310-350 of Seq ID No 58; 4-25, 37-44, 53-59, 72-78, 86-99, 119-128,197-203, 209-218, 220-226, 233-244, 246-254, 264-271, 277-289, 407-430,437-445, 464-472, 482-488, 503-509 and 308-331 of Seq ID No 59; 4-12,14-43, 52-58 and 43-58 of Seq ID No 60; 4-14, 21-29, 35-49 and 38-50 ofSeq ID No 61; 4-19, 31-37, 58-72, 94-108 and 1-72 of Seq ID No 62, andfragments comprising at least 6, preferably more than 8, especially morethan 10 aa of said sequences. All these fragments individually and eachindependently form a preferred selected aspect of the present invention.

All linear hyperimmune serum reactive fragments of a particular antigenmay be identified by analysing the entire sequence of the proteinantigen by a set of peptides overlapping by 1 amino acid with a lengthof at least 10 amino acids. Subsequently, non-linear epitopes can beidentified by analysis of the protein antigen with hyperimmune serausing the expressed full-length protein or domain polypeptides thereof.Assuming that a distinct domain of a protein is sufficient to form the3D structure independent from the native protein, the analysis of therespective recombinant or synthetically produced domain polypeptide withhyperimmune serum would allow the identification of conformationalepitopes within the individual domains of multi-domain proteins. Forthose antigens where a domain possesses linear as well as conformationalepitopes, competition experiments with peptides corresponding to thelinear epitopes may be used to confirm the presence of conformationalepitopes.

It will be appreciated that the invention also relates to, among others,nucleic acid molecules encoding the aforementioned fragments, nucleicacid molecules that hybridise to nucleic acid molecules encoding thefragments, particularly those that hybridise under stringent conditions,and nucleic acid molecules, such as PCR primers, for amplifying nucleicacid molecules that encode the fragments. In these regards, preferrednucleic acid molecules are those that correspond to the preferredfragments, as discussed above.

The present invention also relates to vectors, which comprise a nucleicacid molecule or nucleic acid molecules of the present invention, hostcells which are genetically engineered with vectors of the invention andthe production of hyperimmune serum reactive antigens and fragmentsthereof by recombinant techniques.

A great variety of expression vectors can be used to express ahyperimmune serum reactive antigen or fragment thereof according to thepresent invention. Generally, any vector suitable to maintain, propagateor express nucleic acids to express a polypeptide in a host may be usedfor expression in this regard. In accordance with this aspect of theinvention the vector may be, for example, a plasmid vector, a single ordouble-stranded phage vector, a single or double-stranded RNA or DNAviral vector. Starting plasmids disclosed herein are either commerciallyavailable, publicly available, or can be constructed from availableplasmids by routine application of well-known, published procedures.Preferred among vectors, in certain respects, are those for expressionof nucleic acid molecules and hyperimmune serum reactive antigens orfragments thereof of the present invention. Nucleic acid constructs inhost cells can be used in a conventional manner to produce the geneproduct encoded by the recombinant sequence. Alternatively, thehyperimmune serum reactive antigens and fragments thereof of theinvention can be synthetically produced by conventional peptidesynthesizers. Mature proteins can be expressed in mammalian cells,yeast, bacteria, or other cells under the control of appropriatepromoters. Cell-free translation systems can also be employed to producesuch proteins using RNAs derived from the DNA construct of the presentinvention.

Host cells can be genetically engineered to incorporate nucleic acidmolecules and express nucleic acid molecules of the present invention.Representative examples of appropriate hosts include bacterial cells,such as staphylococci, streptococci, E. coli, Streptomyces and Bacillussubtillis cells; fungal cells, such as yeast cells and Aspergilluscells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, Hela, C127, 3T3, BHK, 293 and Bowesmelanoma cells; and plant cells.

The invention also provides a process for producing a S. epidermidishyperimmune serum reactive antigen and a fragment thereof comprisingexpressing from the host cell a hyperimmune serum reactive antigen orfragment thereof encoded by the nucleic acid molecules provided by thepresent invention. The invention further provides a process forproducing a cell, which expresses a S. epidermidis hyperimmune serumreactive antigen or a fragment thereof comprising trans-forming ortransfecting a suitable host cell with the vector according to thepresent invention such that the transformed or transfected cellexpresses the polypeptide encoded by the nucleic acid contained in thevector.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N- or C-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, regions may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stability orto facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilize orpurify polypeptides. For example, EP-A-O 42-3, 5, 10, 14, 16, 18, 22-24,27 533 (Canadian counterpart 2045869) discloses fusion proteinscomprising various portions of constant region of immunoglobin moleculestogether with another protein or part thereof. In drug discovery, forexample, proteins have been fused with antibody Fc portions for thepurpose of high-throughout screening assays to identify antagonists. Seefor example, {Bennett, D. et al., 1995} and {Johanson, K. et al., 1995}.

The S. epidermidis hyperimmune serum reactive antigen or a fragmentthereof can be recovered and purified from recombinant cell cultures bywell-known methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography and lectin chromatography.

The hyperimmune serum reactive antigens and fragments thereof accordingto the present invention can be produced by chemical synthesis as wellas by biotechnological means. The latter comprise the transfection ortransformation of a host cell with a vector containing a nucleic acidaccording to the present invention and the cultivation of thetransfected or transformed host cell under conditions, which are knownto the ones skilled in the art. The production method may also comprisea purification step in order to purify or isolate the polypeptide to bemanufactured. In a preferred embodiment the vector is a vector accordingto the present invention.

The hyperimmune serum reactive antigens and fragments thereof accordingto the present invention may be used for the detection of the organismor organisms in a sample containing these or ganisms or polypeptidesderived thereof. Preferably such detection is for diagnosis, morepreferable for the diagnosis of a disease, most preferably for thediagnosis of a diseases related or linked to the presence or abundanceof Gram-positive bacteria, especially bacteria selected from the groupcomprising staphylococci, streptococci and lactococci. More preferably,the microorganisms are selected from the group comprising Staphylococcusaureus and Staphylococcus saprophyticus, especially the microorganism isStaphylococcus epidermidis.

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of thehyperimmune serum reactive antigens and fragments thereof of the presentinvention in cells and tissues, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting overexpression of the polypeptidecompared to normal control tissue samples may be used to detect thepresence of an infection, for example, and to identify the infectingorganism. Assay techniques that can be used to determine levels of apolypeptide, in a sample derived from a host are well known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Among these, ELISAs frequently are preferred. An ELISA assay initiallycomprises preparing an antibody specific to the polypeptide, preferablya monoclonal antibody. In addition, a reporter antibody generally isprepared which binds to the monoclonal antibody. The reporter antibodyis attached to a detectable reagent such as radioactive, fluorescent orenzymatic reagent, such as horseradish peroxidase enzyme.

The hyperimmune serum reactive antigens and fragments thereof accordingto the present invention may also be used for the purpose of or inconnection with an array. More particularly, at least one of thehyperimmune serum reactive antigens and fragments thereof according tothe present invention may be immobilized on a support. Said supporttypically comprises a variety of hyperimmune serum reactive antigens andfragments thereof whereby the variety may be created by using one orseveral of the hyperimmune serum reactive antigens and fragments thereofaccording to the present invention and/or hyperimmune serum reactiveantigens and fragments thereof being different. The characterizingfeature of such array as well as of any array in general is the factthat at a distinct or predefined region or position on said support or asurface thereof, a distinct polypeptide is immobilized. Because of thisany activity at a distinct position or region of an array can becorrelated with a specific polypeptide. The number of differenthyperimmune serum reactive antigens and fragments thereof immobilized ona support may range from as little as 10 to several 1000 differenthyperimmune serum reactive antigens and fragments thereof. The densityof hyperimmune serum reactive antigens and fragments thereof per cm² isin a preferred embodiment as little as 10 peptides/polypeptides per cm²to at least 400 different peptides/polypeptides per cm² and moreparticularly at least 1000 different hyperimmune serum reactive antigensand fragments thereof per cm².

The manufacture of such arrays is known to the one skilled in the artand, for example, described in U.S. Pat. No. 5,744,309. The arraypreferably comprises a planar, porous or non-porous solid support havingat least a first surface. The hyperimmune serum reactive antigens andfragments thereof as disclosed herein, are immobilized on said surface.Preferred support materials are, among others, glass or cellulose. It isalso within the present invention that the array is used for any of thediagnostic applications described herein. Apart from the hyperimmuneserum reactive antigens and fragments thereof according to the presentinvention also the nucleic acid molecules according to the presentinvention may be used for the generation of an array as described above.This applies as well to an array made of antibodies, preferablymonoclonal antibodies as, among others, described herein.

In a further aspect the present invention relates to an antibodydirected to any of the hyperimmune serum reactive antigens and fragmentsthereof, derivatives or fragments thereof according to the presentinvention. The present invention includes, for example, monoclonal andpolyclonal antibodies, chimeric, single chain, and humanized antibodies,as well as Fab fragments, or the product of a Fab expression library. Itis within the present invention that the antibody may be chimeric, i.e.that different parts thereof stem from different species or at least therespective sequences are taken from different species.

Antibodies generated against the hyperimmune serum reactive antigens andfragments thereof corresponding to a sequence of the present inventioncan be obtained by direct injection of the hyperimmune serum reactiveantigens and fragments thereof into an animal or by administering thehyperimmune serum reactive antigens and fragments thereof to an animal,preferably a non-human. The antibody so obtained will then bind thehyperimmune serum reactive antigens and fragments thereof itself. Inthis manner, even a sequence encoding only a fragment of a hyperimmuneserum reactive antigen and fragments thereof can be used to generateantibodies binding the whole native hyperimmune serum reactive antigenand fragments thereof. Such antibodies can then be used to isolate thehyperimmune serum reactive antigens and fragments thereof from tissueexpressing those hyperimmune serum reactive antigens and fragmentsthereof.

For preparation of monoclonal antibodies, any technique known in theart, which provides antibodies produced by continuous cell line culturescan be used. (as described originally in {Kohler, G. et al., 1975}.

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic hyperimmune serum reactive antigens and fragments thereofaccording to this invention. Also, transgenic mice, or other organismssuch as other mammals, may be used to express humanized antibodies toimmunogenic hyperimmune serum reactive antigens and fragments thereofaccording to this invention.

Alternatively, phage display technology or ribosomal display could beutilized to select antibody genes with binding activities towards thehyperimmune serum reactive antigens and fragments thereof either fromrepertoires of PCR amplified v-genes of lymphocytes from humans screenedfor possessing respective target antigens or from naïve libraries{McCafferty, J. et al., 1990}; {Marks, J. et al., 1992}. The affinity ofthese antibodies can also be improved by chain shuffling {Clackson, T.et al., 1991}.

If two antigen binding domains are present, each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the hyperimmune serum reactive antigens and fragmentsthereof or purify the hyperimmune serum reactive antigens and fragmentsthereof of the present invention by attachment of the antibody to asolid support for isolation and/or purification by affinitychromatography.

Thus, among others, antibodies against the hyperimmune serum reactiveantigens and fragments thereof of the present invention may be employedto inhibit and/or treat infections, particularly bacterial infectionsand especially infections arising from S. epidermidis.

Hyperimmune serum reactive antigens and fragments thereof includeantigenically, epitopically or immunologically equivalent derivatives,which form a particular aspect of this invention. The term“antigenically equivalent derivative” as used herein encompasses ahyperimmune serum reactive antigen and fragments thereof or itsequivalent which will be specifically recognized by certain antibodieswhich, when raised to the protein or hyperimmune serum reactive antigenand fragments thereof according to the present invention, interfere withthe interaction between pathogen and mammalian host. The term“immunologically equivalent derivative” as used herein encompasses apeptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe interaction between pathogen and mammalian host.

The hyperimmune serum reactive antigens and fragments thereof, such asan antigenically or immunologically equivalent derivative or a fusionprotein thereof can be used as an antigen to immunize a mouse or otheranimal such as a rat or chicken. The fusion protein may providestability to the hyperimmune serum reactive antigens and fragmentsthereof. The antigen may be associated, for example by conjugation, withan immunogenic carrier protein, for example bovine serum albumin (BSA)or keyhole limpet haemocyanin (KLH). Alternatively, an antigenic peptidecomprising multiple copies of the protein or hyperimmune serum reactiveantigen and fragments thereof, or an antigenically or immunologicallyequivalent hyperimmune serum reactive antigen and fragments thereof, maybe sufficiently antigenic to improve immunogenicity so as to obviate theuse of a carrier.

Preferably the antibody or derivative thereof is modified to make itless immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized”, wherein thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in {Jones, P. et al., 1986} or {Tempest, P. et al., 1991}.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscle, delivery of DNA complexed withspecific protein carriers, coprecipitation of DNA with calciumphosphate, encapsulation of DNA in various forms of liposomes, particlebombardment {Tang, D. et al., 1992}, {Eisenbraun, M. et al., 1993} andin vivo infection using cloned retroviral vectors {Seeger, C. et al.,1984}.

In a further aspect the present invention relates to a peptide bindingto any of the hyperimmune serum reactive antigens and fragments thereofaccording to the present invention, and a method for the manufacture ofsuch peptides whereby the method is characterized by the use of thehyperimmune serum reactive antigens and fragments thereof according tothe present invention and the basic steps are known to the one skilledin the art.

Such peptides may be generated by using methods according to the stateof the art such as phage display or ribosome display. In case of phagedisplay, basically a library of peptides is generated, in form ofphages, and this kind of library is contacted with the target molecule,in the present case a hyperimmune serum reactive antigen and fragmentsthereof according to the present invention. Those peptides binding tothe target molecule are subsequently removed, preferably as a complexwith the target molecule, from the respective reaction. It is known tothe one skilled in the art that the binding characteristics, at least toa certain extent, depend on the particularly realized experimentalset-up such as the salt concentration and the like. After separatingthose peptides binding to the target molecule with a higher affinity ora bigger force, from the non-binding members of the library, andoptionally also after removal of the target molecule from the complex oftarget molecule and peptide, the respective peptide(s) may subsequentlybe characterised. Prior to the characterisation optionally anamplification step is realized such as, e.g. by propagating the peptideencoding phages. The characterisation preferably comprises thesequencing of the target binding peptides. Basically, the peptides arenot limited in their lengths, however, peptides having a length fromabout 8 to 20 amino acids are preferably obtained in the respectivemethods. The size of the libraries may be about 102 to 1018, preferably108 to 1015 different peptides, however, is not limited thereto.

A particular form of target binding hyperimmune serum reactive antigensand fragments thereof are the so-called “anticalines” which are, amongothers, described in German patent application DE 197 42 706.

In a further aspect the present invention relates to functional nucleicacids interacting with any of the hyperimmune serum reactive antigensand fragments thereof according to the present invention, and a methodfor the manufacture of such functional nucleic acids whereby the methodis characterized by the use of the hyperimmune serum reactive antigensand fragments thereof α-cording to the present invention and the basicsteps are known to the one skilled in the art. The functional nucleicacids are preferably aptamers and spiegelmers.

Aptamers are D-nucleic acids, which are either single stranded or doublestranded and which specifically interact with a target molecule. Themanufacture or selection of aptamers is, e.g., described in Europeanpatent EP 0 533 838. Basically the following steps are realized. First,a mixture of nucleic acids, i.e. potential aptamers, is provided wherebyeach nucleic acid typically comprises a segment of several, preferablyat least eight subsequent randomised nucleotides. This mixture issubsequently contacted with the target molecule whereby the nucleicacid(s) bind to the target molecule, such as based on an increasedaffinity towards the target or with a bigger force thereto, compared tothe candidate mixture. The binding nucleic acid(s) are/is subsequentlyseparated from the remainder of the mixture. Optionally, the thusobtained nucleic acid(s) is amplified using, e.g. polymerase chainreaction. These steps may be repeated several times giving at the end amixture having an increased ratio of nucleic acids specifically bindingto the target from which the final binding nucleic acid is thenoptionally selected. These specifically binding nucleic acid(s) arereferred to as aptamers. It is obvious that at any stage of the methodfor the generation or identification of the aptamers samples of themixture of individual nucleic acids may be taken to determine thesequence thereof using standard techniques. It is within the presentinvention that the aptamers may be stabilized such as, e.g., byintroducing defined chemical groups which are known to the one skilledin the art of generating aptamers. Such modification may for examplereside in the introduction of an amino group at the 2′-position of thesugar moiety of the nucleotides. Aptamers are currently used astherapeutical agents. However, it is also within the present inventionthat the thus selected or generated aptamers may be used for targetvalidation and/or as lead substance for the development of medicaments,preferably of medicaments based on small molecules. This is actuallydone by a competition assay whereby the specific interaction between thetarget molecule and the aptamer is inhibited by a candidate drug wherebyupon replacement of the aptamer from the complex of target and aptamerit may be assumed that the respective drug candidate allows a specificinhibition of the interaction between target and aptamer, and if theinteraction is specific, said candidate drug will, at least inprinciple, be suitable to block the target and thus decrease itsbiological availability or activity in a respective system comprisingsuch target. The thus obtained small molecule may then be subject tofurther derivatisation and modification to optimise its physical,chemical, biological and/or medical characteristics such as toxicity,specificity, biodegradability and bioavailability.

Spiegelmers and their generation or manufacture is based on a similarprinciple. The manufacture of spiegelmers is described in internationalpatent application WO 98/08856. Spiegelmers are L-nucleic acids, whichmeans that they are composed of L-nucleotides rather than D-nucleotidesas aptamers are. Spiegelmers are characterized by the fact that theyhave a very high stability in biological systems and, comparable toaptamers, specifically interact with the target molecule against whichthey are directed. In the process of generating spiegelmers, aheterogeonous population of D-nucleic acids is created and thispopulation is contacted with the optical antipode of the targetmolecule, in the present case for example with the D-enantiomer of thenaturally occurring L-enantiomer of the hyperimmune serum reactiveantigens and fragments thereof according to the present invention.Subsequently, those D-nucleic acids are separated which do not interactwith the optical antipode of the target molecule. But those D-nucleicacids interacting with the optical antipode of the target molecule areseparated, optionally identified and/or sequenced and subsequently thecorresponding L-nucleic acids are synthesized based on the nucleic acidsequence information obtained from the D-nucleic acids. These L-nucleicacids, which are identical in terms of sequence with the aforementionedD-nucleic acids interacting with the optical antipode of the targetmolecule, will specifically interact with the naturally occurring targetmolecule rather than with the optical antipode thereof. Similar to themethod for the generation of aptamers it is also possible to repeat thevarious steps several times and thus to enrich those nucleic acidsspecifically interacting with the optical antipode of the targetmolecule.

In a further aspect the present invention relates to functional nucleicacids interacting with any of the nucleic acid molecules according tothe present invention, and a method for the manufacture of suchfunctional nucleic acids whereby the method is characterized by the useof the nucleic acid molecules and their respective sequences accordingto the present invention and the basic steps are known to the oneskilled in the art. The functional nucleic acids are preferablyribozymes, antisense oligonucleotides and siRNA.

Ribozymes are catalytically active nucleic acids, which preferablyconsist of RNA, which basically comprises two moieties. The first moietyshows a catalytic activity whereas the second moiety is responsible forthe specific interaction with the target nucleic acid, in the presentcase the nucleic acid coding for the hyperimmune serum reactive antigensand fragments thereof according to the present invention. Uponinteraction between the target nucleic acid and the second moiety of theribozyme, typically by hybridisation and Watson-Crick base pairing ofessentially complementary stretches of bases on the two hybridisingstrands, the catalytically active moiety may become active which meansthat it catalyses, either intramolecularly or intermolecularly, thetarget nucleic acid in case the catalytic activity of the ribozyme is aphosphodiesterase activity. Subsequently, there may be a furtherdegradation of the target nucleic acid, which in the end results in thedegradation of the target nucleic acid as well as the protein derivedfrom the said target nucleic acid. Ribozymes, their use and designprinciples are known to the one skilled in the art, and, for exampledescribed in {Doherty, E. et al., 2001} and {Lewin, A. et al., 2001}.

The activity and design of antisense oligonucleotides for themanufacture of a medicament and as a diagnostic agent, respectively, isbased on a similar mode of action. Basically, antisense oligonucleotideshybridise based on base complementarity, with a target RNA, preferablywith a mRNA, thereby activating RNase H. RNase H is activated by bothphosphodiester and phosphorothioate-coupled DNA. Phosphodiester-coupledDNA, however, is rapidly degraded by cellular nucleases with theexception of phosphorothioate-coupled DNA. These resistant,non-naturally occurring DNA derivatives do not inhibit RNase H uponhybridisation with RNA. In other words, antisense polynucleotides areonly effective as DNA RNA hybride complexes. Examples for this kind ofantisense oligonucleotides are described, among others, in U.S. Pat. No.5,849,902 and U.S. Pat. No. 5,989,912. In other words, based on thenucleic acid sequence of the target molecule which in the present caseare the nucleic acid molecules for the hyperimmune serum reactiveantigens and fragments thereof according to the present invention,either from the target protein from which a respective nucleic acidsequence may in principle be deduced, or by knowing the nucleic acidsequence as such, particularly the mRNA, suitable antisenseoligonucleotides may be designed base on the principle of basecomplementarity.

Particularly preferred are antisense-oligonucleotides, which have ashort stretch of phosphorothioate DNA (3 to 9 bases). A minimum of 3 DNAbases is required for activation of bacterial RNase H and a minimum of 5bases is required for mammalian RNase H activation. In these chimericoligonucleotides there is a central region that forms a substrate forRNase H that is flanked by hybridising “arms” comprised of modifiednucleotides that do not form substrates for RNase H. The hybridisingarms of the chimeric oligonucleotides may be modified such as by2′-O-methyl or 2′-fluoro. Alternative approaches used methylphosphonateor phosphoramidate linkages in said arms. Further embodiments of theantisense oligonucleotide useful in the practice of the presentinvention are P-methoxyoligonucleotides, partialPmethoxyoligodeoxyribonucleotides or P-methoxyoligonucleotides.

Of particular relevance and usefulness for the present invention arethose antisense oligonucleotides as more particularly described in theabove two mentioned US patents. These oligonucleotides contain nonaturally occurring 5′ 3′-linked nucleotides. Rather theoligonucleotides have two types of nucleotides:2′-deoxyphosphorothioate, which activate RNase H, and 2′-modifiednucleotides, which do not. The linkages between the 2′-modifiednucleotides can be phosphodiesters, phosphorothioate orP-ethoxyphosphodiester. Activation of RNase H is accomplished by acontiguous RNase H-activating region, which contains between 3 and 52′-deoxyphosphorothioate nucleotides to activate bacterial RNase H andbetween 5 and 10 2′-deoxyphosphorothioate nucleotides to activateeukaryotic and, particularly, mammalian RNase H. Protection fromdegradation is accomplished by making the 5′ and 3′ terminal baseshighly nuclease resistant and, optionally, by placing a 3′ terminalblocking group.

More particularly, the antisense oligonucleotide comprises a 5′ terminusand a 3′ terminus; and from position 11 to 59 5′ 3′-linked nucleotidesindependently selected from the group consisting of 2′-modifiedphosphodiester nucleotides and 2′-modified P-alkyloxyphosphotriesternucleotides; and wherein the 5′-terminal nucleoside is attached to anRNase H-activating region of between three and ten contiguousphosphorothioate-linked deoxyribonucleotides, and wherein the3′-terminus of said oligonucleotide is selected from the groupconsisting of an inverted deoxyribonucleotide, a contiguous stretch ofone to three phosphorothioate 2′-modified ribonucleotides, a biotingroup and a P-alkyloxyphosphotriester nucleotide.

Also an antisense oligonucleotide may be used wherein not the 5′terminal nucleoside is attached to an RNase H-activating region but the3′ terminal nucleoside as specified above. Also, the 5′ terminus isselected from the particular group rather than the 3′ terminus of saidoligonucleotide.

The nucleic acids as well as the hyperimmune serum reactive antigens andfragments thereof according to the present invention may be used as orfor the manufacture of pharmaceutical compositions, especially vaccines.Preferably such pharmaceutical composition, preferably vaccine is forthe prevention or treatment of diseases caused by, related to orassociated with S. epidermidis. In so far another aspect of theinvention relates to a method for inducing an immunological response inan individual, particularly a mammal, which comprises inoculating theindividual with the hyperimmune serum reactive antigens and fragmentsthereof of the invention, or a fragment or variant thereof, adequate toproduce antibodies to protect said individual from infection,particularly Staphylococcus infection and most particularly S.epidermidis infections.

Yet another aspect of the invention relates to a method of inducing animmunological response in an individual which comprises, through genetherapy or otherwise, delivering a nucleic acid functionally encodinghyperimmune serum reactive antigens and fragments thereof, or a fragmentor a variant thereof, for expressing the hyperimmune serum reactiveantigens and fragments thereof, or a fragment or a variant thereof invivo in order to induce an immunological response to produce antibodiesor a cell mediated T cell response, either cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether thatdisease is already established within the individual or not. One way ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into a host capable of having inducedwithin it an immunological response, induces an immunological responsein such host, wherein the composition comprises recombinant DNA whichcodes for and expresses an antigen of the hyperimmune serum reactiveantigens and fragments thereof of the present invention. Theimmunological response may be used therapeutically or prophylacticallyand may take the form of antibody immunity or cellular immunity such asthat arising from CTL or CD4+ T cells.

The hyperimmune serum reactive antigens and fragments thereof of theinvention or a fragment thereof may be fused with a co-protein which maynot by itself produce antibodies, but is capable of stabilizing thefirst protein and producing a fused protein which will have immunogenicand protective properties. This fused recombinant protein preferablyfurther comprises an antigenic co-protein, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largecoproteins which solubilise the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

Also, provided by this invention are methods using the described nucleicacid molecule or particular fragments thereof in such geneticimmunization experiments in animal models of infection with S.epidermidis. Such fragments will be particularly useful for identifyingprotein epitopes able to provoke a prophylactic or therapeutic immuneresponse. This approach can allow for the subsequent preparation ofmonoclonal antibodies of particular value from the requisite organ ofthe animal successfully resisting or clearing infection for thedevelopment of prophylactic agents or therapeutic treatments of S.epidermidis infection in mammals, particularly humans.

The hyperimmune serum reactive antigens and fragments thereof may beused as an antigen for vaccination of a host to produce specificantibodies which protect against invasion of bacteria, for example byblocking adherence of bacteria to damaged tissue. Examples of tissuedamage include wounds in skin or connective tissue caused e.g. bymechanical, chemical or thermal damage or by implantation of indwellingdevices, or wounds in the mucous membranes, such as the mouth, mammaryglands, urethra or vagina.

The present invention also includes a vaccine formulation, whichcomprises the immunogenic recombinant protein together with a suitablecarrier. Since the protein may be broken down in the stomach, it ispreferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous,intradermal intranasal or transdermal. Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with thebodily fluid, preferably the blood, of the individual; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampoules and vials, and maybe stored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The vaccine formulationmay also include adjuvant systems for enhancing the immunogenicity ofthe formulation, such as oil-in-water systems and other systems known inthe art. The dosage will depend on the specific activity of the vaccineand can be readily determined by routine experimentation.

According to another aspect, the present invention relates to apharmaceutical composition comprising such a hyperimmune serum-reactiveantigen or a fragment thereof as provided in the present invention forS. epidermidis. Such a pharmaceutical composition may comprise one ormore hyperimmune serum reactive antigens or fragments thereof against S.epidermidis. Optionally, such S. epidermidis hyperimmune serum reactiveantigens or fragments thereof may also be combined with antigens againstother pathogens in a combination pharmaceutical composition. Preferably,said pharmaceutical composition is a vaccine for preventing or treatingan infection caused by S. epidermidis and/or other pathogens againstwhich the antigens have been included in the vaccine.

According to a further aspect, the present invention relates to apharmaceutical composition comprising a nucleic acid molecule encoding ahyperimmune serum-reactive antigen or a fragment thereof as identifiedabove for S. epidermidis. Such a pharmaceutical composition may compriseone or more nucleic acid molecules encoding hyperimmune serum reactiveantigens or fragments thereof against S. epidermidis. Optionally, suchS. epidermidis nucleic acid molecules encoding hyperimmune serumreactive antigens or fragments thereof may also be combined with nucleicacid molecules encoding antigens against other pathogens in acombination pharmaceutical composition. Preferably, said pharmaceuticalcomposition is a vaccine for preventing or treating an infection causedby S. epidermidis and/or other pathogens against which the antigens havebeen included in the vaccine.

The pharmaceutical composition may contain any suitable auxiliarysubstances, such as buffer substances, stabilisers or further activeingredients, especially ingredients known in connection ofpharmaceutical composition and/or vaccine production.

A preferable carrier/or excipient for the hyperimmune serum-reactiveantigens, fragments thereof or a coding nucleic acid molecule thereofaccording to the present invention is an immunostimulatory compound forfurther stimulating the immune response to the given hyperimmuneserum-reactive antigen, fragment thereof or a coding nucleic acidmolecule thereof. Preferably the immunostimulatory compound in thepharmaceutical preparation according to the present invention isselected from the group of polycationic substances, especiallypolycationic peptides, immunostimulatory nucleic acids molecules,preferably immunostimulatory deoxynucleotides, alum, Freund's completeadjuvants, Freund's incomplete adjuvants, neuroactive compounds,especially human growth hormone, or combinations thereof.

It is also within the scope of the present invention that thepharmaceutical composition, especially vaccine, comprises apart from thehyperimmune serum reactive antigens, fragments thereof and/or codingnucleic acid molecules thereof according to the present invention othercompounds which are biologically or pharmaceutically active. Preferably,the vaccine composition comprises at least one polycationic peptide. Thepolycationic compound(s) to be used according to the present inventionmay be any polycationic compound, which shows the characteristic effectsaccording to the WO 97/30721. Preferred polycationic compounds areselected from basic polypeptides, organic polycations, basic polyaminoacids or mixtures thereof. These polyamino acids should have a chainlength of at least 4 amino acid residues (WO 97/30721). Especiallypreferred are substances like polylysine, polyarginine and polypeptidescontaining more than 20%, especially more than 50% of basic amino acidsin a range of more than 8, especially more than 20, amino acid residuesor mixtures thereof. Other preferred polycations and theirpharmaceutical compositions are described in WO 97/30721 (e.g.polyethyleneimine) and WO 99/38528. Preferably these polypeptidescontain between 20 and 500 amino acid residues, especially between 30and 200 residues.

These polycationic compounds may be produced chemically or recombinantlyor may be derived from natural sources.

Cationic (poly)peptides may also be anti-microbial with properties asreviewed in {Ganz, T., 1999}. These (poly)peptides may be of prokaryoticor animal or plant origin or may be produced chemically or recombinantly(WO 02/13857). Peptides may also belong to the class of defensins (WO02/13857). Sequences of such peptides can be, for example, found in theAntimicrobial Sequences Database available on the World Wide Web underthe following internet address:

-   -   bbcm.univ.trieste.it/˜tossi/pag2.html

Such host defence peptides or defensives are also a preferred form ofthe polycationic polymer according to the present invention. Generally,a compound allowing as an end product activation (or down-regulation) ofthe adaptive immune system, preferably mediated by APCs (includingdendritic cells) is used as polycationic polymer.

Especially preferred for use as polycationic substances in the presentinvention are cathelicidin derived antimicrobial peptides or derivativesthereof (International patent application WO 02/13857, incorporatedherein by reference), especially antimicrobial peptides derived frommammalian cathelicidin, preferably from human, bovine or mouse.

Polycationic compounds derived from natural sources include HW-REV orHIV-TAT (derived cationic peptides, antennapedia peptides, chitosan orother derivatives of chitin) or other peptides derived from thesepeptides or proteins by biochemical or recombinant production. Otherpreferred polycationic compounds are cathelin or related or derivedsubstances from cathelin. For example, mouse cathelin is a peptide,which has the amino acid sequenceNH₂-RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE-COOH (SEQ ID NO:64). Related orderived cathelin substances contain the whole or parts of the cathelinsequence with at least 15-20 amino acid residues. Derivations mayinclude the substitution or modification of the natural amino acids byamino acids, which are not among the 20 standard amino acids. Moreover,further cationic residues may be introduced into such cathelinmolecules. These cathelin molecules are preferred to be combined withthe antigen. These cathelin molecules surprisingly have turned out to bealso effective as an adjuvant for an antigen without the addition offurther adjuvants. It is therefore possible to use such cathelinmolecules as efficient adjuvants in vaccine formulations with or withoutfurther immunoactivating substances.

Another preferred polycationic substance to be used according to thepresent invention is a synthetic peptide containing at least 2KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids(International patent application WO 02/32451, incorporated herein byreference).

The pharmaceutical composition of the present invention may furthercomprise immunostimulatory nucleic acid(s). Immunostimulatory nucleicacids are e.g. neutral or artificial CpG containing nucleic acids, shortstretches of nucleic acids derived from non-vertebrates or in form ofshort oligonucleotides (ODNs) containing non-methylated cytosine-guaninedi-nucleotides (CpG) in a certain base context (e.g. described in WO96/02555). Alternatively, also nucleic acids based on inosine andcytidine as e.g. described in the WO 01/93903, or deoxynucleic acidscontaining deoxy-inosine and/or deoxyuridine residues (described in WO01/93905 and PCT/EP 02/05448, incorporated herein by reference) maypreferably be used as immunostimulatory nucleic acids for the presentinvention. Preferably, the mixtures of different immunostimulatorynucleic acids may be used according to the present invention.

It is also within the present invention that any of the aforementionedpolycationic compounds is combined with any of the immunostimulatorynucleic acids as aforementioned. Preferably, such combinations areaccording to the ones as described in WO 01/93905, WO 02/32451, WO01/54720, WO 01/93903, WO 02/13857 and PCT/EP 02/05448 and the Austrianpatent application A 1924/2001, incorporated herein by reference.

In addition or alternatively such vaccine composition may comprise apartfrom the hyperimmune serum reactive antigens and fragments thereof, andthe coding nucleic acid molecules thereof according to the presentinvention a neuroactive compound. Preferably, the neuroactive compoundis human growth factor as, e.g. described in WO 01/24822. Alsopreferably, the neuroactive compound is combined with any of thepolycationic compounds and/or immunostimulatory nucleic acids asafore-mentioned.

In a further aspect the present invention is related to a pharmaceuticalcomposition. Such pharmaceutical composition is, for example, thevaccine described herein. Also a pharmaceutical composition is apharmaceutical composition which comprises any of the followingcompounds or combinations thereof: the nucleic acid molecules accordingto the present invention, the hyperimmune serum reactive antigens andfragments thereof according to the present invention, the vectoraccording to the present invention, the cells according to the presentinvention, the antibody according to the present invention, thefunctional nucleic acids according to the present invention and thebinding peptides such as the anticalines according to the presentinvention, any agonists and antagonists screened as described herein. Inconnection therewith any of these compounds may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa hyperimmune serum reactive antigen and fragments thereof of theinvention and a pharmaceutically acceptable carrier or excipient. Suchcarriers may include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical application,for example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.05-5 μg antigen/per kg of body weight,and such dose is preferably administered 1-3 times and with an intervalof 1-3 weeks.

With the indicated dose range, no adverse toxicological effects shouldbe observed with the compounds of the invention, which would precludetheir administration to suitable individuals.

In a further embodiment the present invention relates to diagnostic andpharmaceutical packs and kits comprising one or more containers filledwith one or more of the ingredients of the afore-mentioned compositionsof the invention. The ingredient(s) can be present in a useful amount,dosage, formulation or combination. Associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, reflecting approval by the agency of the manufacture, use orsale of the product for human administration.

In connection with the present invention any disease related use asdisclosed herein such as, e.g. use of the pharmaceutical composition orvaccine, is particularly a disease or diseased condition which is causedby, linked or associated with Staphylococci, more preferably, S.epidermidis. In connection therewith it is to be noted that S.epidermidis comprises several strains including those disclosed herein.A disease related, caused or associated with the bacterial infection tobe prevented and/or treated according to the present invention includesbesides other diseases mostly those related to the presence of foreignbodies and the use of devices, such as catheters, cerebrospinal fluidshunt infections, peritonitis and endocarditis in humans.

In a still further embodiment the present invention is related to ascreening method using any of the hyperimmune serum reactive antigens ornucleic acids according to the present invention. Screening methods assuch are known to the one skilled in the art and can be designed suchthat an agonist or an antagonist is screened. Preferably an antagonistis screened which in the present case inhibits or prevents the bindingof any hyperimmune serum reactive antigen and fragment thereof accordingto the present invention to an interaction partner. Such interactionpartner can be a naturally occurring interaction partner or anon-naturally occurring interaction partner.

The invention also provides a method of screening compounds to identifythose, which enhance (agonist) or block (antagonist) the function ofhyperimmune serum reactive antigens and fragments thereof or nucleicacid molecules of the present invention, such as its interaction with abinding molecule. The method of screening may involve high-throughput.

For example, to screen for agonists or antagonists, the interactionpartner of the nucleic acid molecule and nucleic acid, respectively,according to the present invention, maybe a synthetic reaction mix, acellular compartment, such as a membrane, cell envelope or cell wall, ora preparation of any thereof, may be prepared from a cell that expressesa molecule that binds to the hyperimmune serum reactive antigens andfragments thereof of the present invention. The preparation is incubatedwith labelled hyperimmune serum reactive antigens and fragments thereofin the absence or the presence of a candidate molecule, which may be anagonist or antagonist. The ability of the candidate molecule to bind thebinding molecule is reflected in decreased binding of the labelledligand. Molecules which bind gratuitously, i.e., without inducing thefunctional effects of the hyperimmune serum reactive antigens andfragments thereof, are most likely to be good antagonists. Moleculesthat bind well and elicit functional effects that are the same as orclosely related to the hyperimmune serum reactive antigens and fragmentsthereof are good agonists.

The functional effects of potential agonists and antagonists may bemeasured, for instance, by determining the activity of a reporter systemfollowing interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that of thehyperimmune serum reactive antigens and fragments thereof of the presentinvention or molecules that elicit the same effects as the hyperimmuneserum reactive antigens and fragments thereof. Reporter systems that maybe useful in this regard include but are not limited to colorimetriclabelled substrate converted into product, a reporter gene that isresponsive to changes in the functional activity of the hyperimmuneserum reactive antigens and fragments thereof, and binding assays knownin the art.

Another example of an assay for antagonists is a competitive assay thatcombines the hyperimmune serum reactive antigens and fragments thereofof the present invention and a potential antagonist with membrane-boundbinding molecules, recombinant binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. The hyperimmune serum reactiveantigens and fragments thereof can be labelled such as by radioactivityor a colorimetric compound, such that the molecule number of hyperimmuneserum reactive antigens and fragments thereof bound to a bindingmolecule or converted to product can be determined accurately to assessthe effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a hyperimmune serum reactiveantigen and fragments thereof of the invention and thereby inhibit orextinguish its activity. Potential antagonists also may be small organicmolecules, a peptide, a polypeptide such as a closely related protein orantibody that binds to the same sites on a binding molecule withoutinducing functional activity of the hyperimmune serum reactive antigensand fragments thereof of the invention.

Potential antagonists include a small molecule, which binds to andoccupies the binding site of the hyperimmune serum reactive antigens andfragments thereof thereby preventing binding to cellular bindingmolecules, such that normal biological activity is prevented. Examplesof small molecules include but are not limited to small organicmolecules, peptides or peptide-like molecules.

Other potential antagonists include antisense molecules (see {Okano, H.et al., 1991}; OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENEEXPRESSION; CRC Press, Boca Ration, Fla. (1988), for a description ofthese molecules).

Preferred potential antagonists include derivatives of the hyperimmuneserum reactive antigens and fragments thereof of the invention.

As used herein the activity of a hyperimmune serum reactive antigen andfragment thereof according to the present invention is its capability tobind to any of its interaction partner or the extent of such capabilityto bind to its or any interaction partner.

In a particular aspect, the invention provides the use of thehyperimmune serum reactive antigens and fragments thereof, nucleic acidmolecules or inhibitors of the invention to interfere with the initialphysical interaction between a pathogen and mammalian host responsiblefor sequelae of infection. In particular the molecules of the inventionmay be used: i) in the prevention of adhesion of S. epidermidis tomammalian extracellular matrix proteins on in-dwelling devices or toextracellular matrix proteins in wounds; ii) to block protein mediatedmammalian cell invasion by, for example, initiating phosphorylation ofmammalian tyrosine kinases {Rosenshine, I. et al., 1992} to blockbacterial adhesion between mammalian extracellular matrix proteins andbacterial proteins which mediate tissue damage; iv) to block the normalprogression of pathogenesis in infections initiated other than by theimplantation of in-dwelling devices or by other surgical techniques.

Each of the DNA coding sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein upon expression can be used as a target for the screening ofantibacterial drugs. Additionally, the DNA sequences encoding the aminoterminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct antisense sequences to control the expression of the codingsequence of interest.

The antagonists and agonists may be employed, for instance, to inhibitdiseases arising from infection with Staphylococcus, especially S.epidermidis, such as sepsis.

In a still further aspect the present invention is related to anaffinity device such affinity device comprises as least a supportmaterial and any of the hyperimmune serum reactive antigens andfragments thereof according to the present invention, which is attachedto the support material. Because of the specificity of the hyperimmuneserum reactive antigens and fragments thereof according to the presentinvention for their target cells or target molecules or theirinteraction partners, the hyperimmune serum reactive antigens andfragments thereof allow a selective removal of their interactionpartner(s) from any kind of sample applied to the support materialprovided that the conditions for binding are met. The sample may be abiological or medical sample, including but not limited to, fermentationbroth, cell debris, cell preparation, tissue preparation, organpreparation, blood, urine, lymph liquid, liquor and the like.

The hyperimmune serum reactive antigens and fragments thereof may beattached to the matrix in a covalent or non-covalent manner. Suitablesupport material is known to the one skilled in the art and can beselected from the group comprising cellulose, silicon, glass, aluminium,paramagnetic beads, starch and dextrane.

The present invention is further illustrated by the following figures,examples and the sequence listing from which further features,embodiments and advantages may be taken. It is to be understood that thepresent examples are given by way of illustration only and not by way oflimitation of the disclosure.

In connection with the present invention

FIG. 1 shows the characterization of the selected human high titre seraspecific for S. epidermidis.

FIG. 2 shows the characterization of the small fragment genomic library,LSE-70, from Staphylococcus epidermidis RP62A.

FIG. 3 shows the selection of bacterial cells by MACS using biotinylatedhuman IgGs.

FIG. 4 shows an example for the gene distribution study with theidentified antigens.

Table 1 shows the summary of the screens performed with genomic S.epidermidis libraries and human serum and the gene distribution data forselected antigens.

The figures to which it might be referred to in the specification aredescribed in the following in more details.

FIG. 1 shows the characterization and selection of human serum samplesfor identification of S. epidermidis antigens. (A) ELISA: Total anti-S.epidermidis IgGs were measured by standard ELISA using total bacteriallysate as coating antigen at two different serum dilutions. Five sera(EP.1-5) were selected from a serum collection obtained from patientswith S. epidermidis peritonitis. C, control serum from a patient withunrelated infection. (B) Immunoblot analysis: Selected high titer serawere characterized by immunoblotting using total bacterial lysatesprepared from eight different S. epidermidis clinical isolates (lanes1-8), as well as from S. epidermidis strain RP62A (lane C). In eachlane, ˜20 μg total lysate proteins extracted from bacteria grown in BHImedium overnight were loaded. A representative immunoblot is shown forthe EP.4 serum. The membrane was incubated with EP.4 serum at a dilutionof 5,000 and developed with anti-human IgG secondary reagent. Mw,Protein standards (kDa).

FIG. 2A shows the fragment size distribution of the Staphylococcusepidermidis RP62A small fragment genomic library, LSE-70. Aftersequencing 572 randomly selected clones, sequences were trimmed toeliminate vector residues and the numbers of clones with various genomicfragment sizes were plotted. (B) Graphic illustration of thedistribution of the same set of randomly sequenced clones of LSE-70 overthe S. epidermidis chromosome. Circles indicate matching sequences toannotated ORFs in +/+ and +/− orientation. Rectangles represent fullymatched clones to non-coding chromosomal sequences in +/+ and +/−orientation. Diamonds position the best match of all chimeric clonesequences. Numeric distances in base pairs are indicated over thecircular genome for orientation. Partitioning of various clone setswithin the library is given in numbers and percentage at the bottom ofthe figure.

FIG. 3A shows the MACS selection with biotinylated human IgGs. TheLSE-70 library in pMAL9.1 was screened with 10 μg biotinylated, humanserum (P15-IgG) in the first and second selection round. As negativecontrol, no serum was added to the library cells for screening. Numberof cells selected after the 1st and 2nd elution are shown for eachselection round. FIG. 3B shows the reactivity of specific clones (1-26)isolated by bacterial surface display as analysed by Western blotanalysis with the human serum (P15-IgG) used for selection by MACS at adilution of 1:3,000. As a loading control the same blot was alsoanalysed with antibodies directed against the platform protein LamB at adilution of 1:5,000. LB, Extract from a clone expressing LamB withoutforeign peptide insert.

FIG. 4 shows the PCR analysis for the gene distribution of ORF1163 withthe respective oligonucleotides. The predicted size of the PCR fragmentsis approximately 1,000 bp. The 31 coagulase negative Staphylococcus and11 S. epidermidis strains used for analysis are marked in the figure; N,no genomic DNA added; P, genomic DNA from S. epidermidis RP62A, whichserved as template for library construction.

Table 1: Immunogenic proteins identified by bacterial surface display.

A, LSE-70 library in lamB with P15-IgG (804), B, LSE-150 library in fhuAwith P15-IgG (826), C, LSA-300 library in fhuA with P15-IgG (729), *,prediction of antigenic sequences longer than 5 amino acids wasperformed with the program ANTIGENIC {Kolaskar, A. et al., 1990}. §,Forty-two coagulase negative Staphylococcus or S. epidermidis strainswere tested by PCR with oligonucleotides specific for the genes encodingrelevant antigens. Since 6 of the 31 CNS strains were negative for allgenes analysed, we eliminated these data from the summary, because thesestrains are most likely not closely related to S. epidermidis.

EXAMPLES Example 1 Preparation of Antibodies from Human SerumExperimental Procedures Peptide Synthesis

Peptides were synthesized in small scale (4 mg resin; up to 288 inparallel) using standard F-moc chemistry on a Rink amide resin (PepChem,Tübingen, Germany) using a Syroll synthesizer (Multisyntech, Witten,Germany). After the sequence was assembled, peptides were elongated withFmoc-epsilon-aminohexanoic acid (as a linker) and biotin (Sigma, St.Louis, Mo.; activated like a normal amino acid). Peptides were cleavedoff the resin with 93% TFA, 5% triethylsilane, and 2% water for onehour. Peptides were dried under vacuum and freeze dried three times fromacetonitrile/water (1:1). The presence of the correct mass was verifiedby mass spectrometry on a Reflex III MALDI-TOF (Bruker, Bremen Germany).The peptides were used without further purification.

Enzyme linked immune assay (ELISA).

For serum characterization: ELISA plates (Maxisorb, Millipore) werecoated with 5-10 μg/ml total protein diluted in coating buffer (0.1Msodium carbonate pH 9.2). Three dilutions of sera (2,000×, 10,000×,50,000×) were made in PBS-BSA.

For peptide serology: Biotin-labeled peptides were coating onStreptavidin ELISA plates (EXICON) at 10 μg/ml concentration accordingto the manufacturer's instructions. Sera were tested at two dilutions,200× and 1,000×.

Highly specific Horse Radish Peroxidase (HRP)-conjugated anti-human IgGor anti-human IgA secondary antibodies (Southern Biotech) were usedaccording to the manufacturers' recommendations (dilution: 1,000×).Antigen-antibody complexes were quantified by measuring the conversionof the substrate (ABTS) to colored product based on OD405 nm readings inan automated ELISA reader (TECAN SUNRISE). Following manual coating,peptide plates were processed and analyzed by the Gemini 160 ELISA robot(TECAN) with a built-in reader (GENIOS, TECAN).

Immunoblotting

Total bacterial lysate and culture supernatant samples were preparedfrom in vitro grown S. epidermidis RP62A. 10 to 25 μg total protein/lanewas separated by SDS-PAGE using the BioRad Mini-Protean 3 Cellelectrophoresis system and proteins transferred to nitrocellulosemembrane (ECL, Amersham Pharmacia). After overnight blocking in 5% milk,antisera at 2,000× dilution were added, and HRPO labeled anti-mouse IgGwas used for detection.

Preparation of Bacterial Antigen Extracts

Total bacterial lysate: Bacteria were lysed by repeated freeze-thawcycles: incubation on dry ice/ethanol-mixture until frozen (1 min), thenthawed at 370C (5 min): repeated 3 times. This was followed bysonication and collection of supernatant by centrifugation (3,500 rpm,15 min, 40C).

Culture supernatant: After removal of bacteria, the supernatant ofovernight grown bacterial cultures was precipitated with ice-coldethanol (100%): 1 part supernatant/3 parts ethanol incubated o/n at −20°C. Precipitates were collected by centrifugation (2,600 g, for 15 min)and dried. Dry pellets were dissolved either in PBS for ELISA, or inurea and SDS-sample buffer for SDS-PAGE and immunoblotting. The proteinconcentration of samples was determined by Bradford assay.

Purification of antibodies for genomic screening. Five sera from thepatient group were selected based on the overall anti-staphylococcaltiters for a serum pool used in the screening procedure. Antibodiesagainst E. coli proteins were removed by incubating the heat-inactivatedsera with whole cell E. coli cells (DHSalpha, transformed with pHIE11,grown under the same condition as used for bacterial surface display).Highly enriched preparations of IgGs from the pooled, depleted sera weregenerated by protein G affinity chromatography, according to themanufacturer's instructions (UltraLink Immobilized Protein G, Pierce).IgA antibodies were purified also by affinity chromatography usingbiotin-labeled anti-human IgA (Southern Biotech) immobilized onStreptavidin-agarose (GIBCO BRL). The efficiency of depletion andpurification was checked by SDS-PAGE, Western blotting, ELISA andprotein concentration measurements.

The antibodies produced against S. epidermidis by the human immunesystem and present in human sera are indicative of the in vivoexpression of the antigenic proteins and their immunogenicity. Thesemolecules are essential for the identification of individual antigens inthe approach as described in the present invention, which is based onthe interaction of the specific anti-staphylococcal antibodies and thecorresponding S. epidermidis peptides or proteins. To gain access torelevant antibody repertoires, human sera were collected fromconvalescent patients with S. epidermidis infections, namelyperitonitis.

The sera were characterized for anti-S. epidermidis antibodies by aseries of ELISA and immunoblotting assays. Bacterial lysate proteinsprepared from S. epidermidis RP62A cultured overnight (stationary phase)in BHI (Brain Heart Infusion) growth medium have been used asstaphylococcal antigens. Both IgG and IgA antibody levels weredetermined. Five sera having the highest antibody levels were pooled,and IgG prepared for use in bacterial surface display in order toidentify antigenic proteins.

The titers were compared at given dilutions where the response waslinear. Sera were ranked based on the reactivity against multiplestaphylococcal components, and the highest ones were selected forfurther analysis by immunoblotting (FIG. 1). This extensive antibodycharacterization approach has led to the unambiguous identification ofanti-staphylococcal hyperimmune sera.

Example 2 Generation of Highly Random, Frame-Selected, Small-Fragment,Genomic DNA Libraries of Staphylococcus epidermidis ExperimentalProcedures

Preparation of staphylococcal genomic DNA. 50 ml BHI medium wasinoculated with S. epidermidis RP62A bacteria from a frozen stab andgrown with aeration and shaking for 18 h at 37° C. The culture was thenharvested, centrifuged with 1,600×g for 15 min and the supernatant wasremoved. Bacterial pellets were washed 3× with PBS and carefullyre-suspended in 0.5 ml of Lysozyme solution (100 mg/ml). 0.1 ml of 10mg/ml heat treated RNase A and 20 U of RNase T1 were added, mixedcarefully and the solution was incubated for 1 h at 37° C. Following theaddition of 0.2 ml of 20% SDS solution and 0.1 ml of Proteinase K (10mg/ml) the tube was incubated overnight at 55° C. ⅓ volume of saturatedNaCl was then added and the solution was incubated for 20 min at 4° C.The extract was pelleted in a microfuge (13,000 rpm) and the supernatanttransferred into a new tube. The solution was extracted withPhOH/CHCl₃/IAA (25:24:1) and with CHCl₃/IAA (24:1). DNA was precipitatedat room temperature by adding 0.6× volume of Isopropanol, spooled fromthe solution with a sterile Pasteur pipette and transferred into tubescontaining 80% ice-cold ethanol. DNA was recovered by centrifuging theprecipitates with 10-12,000×g, then dried on air and dissolved in ddH2O.

Preparation of small genomic DNA fragments. Genomic DNA fragments weremechanically sheared into fragments ranging in size between 150 and 300bp using a cup-horn sonicator (Bandelin Sonoplus UV 2200 sonicatorequipped with a BB5 cup horn, 10 sec. pulses at 100% power output) orinto fragments of size between 50 and 70 bp by mild DNase I treatment(Novagen). It was observed that sonication yielded a much tighterfragment size distribution when breaking the DNA into fragments of the150-300 bp size range. However, despite extensive exposure of the DNA toultrasonic wave-induced hydromechanical shearing force, subsequentdecrease in fragment size could not be efficiently and reproduciblyachieved. Therefore, fragments of 50 to 70 bp in size were obtained bymild DNase I treatment using Novagen's shotgun cleavage kit. A 1:20dilution of DNase I provided with the kit was prepared and the digestionwas performed in the presence of MnCl₂ in a 60 μl volume at 20° C. for 5min to ensure double-stranded cleavage by the enzyme. Reactions werestopped with 2 μl of 0.5 M EDTA and the fragmentation efficiency wasevaluated on a 2% TAE-agarose gel. This treatment resulted in totalfragmentation of genomic DNA into near 50-70 bp fragments. Fragmentswere then blunt-ended twice using T4 DNA Polymerase in the presence of100 μM each of dNTPs to ensure efficient flushing of the ends. Fragmentswere used immediately in ligation reactions or frozen at −20° C. forsubsequent use.

Description of the vectors. The vector pMAL4.31 was constructed on apASK-IBA backbone {Skerra, A., 1994} with the beta-lactamase (bla) geneexchanged with the Kanamycin resistance gene. In addition the bla genewas cloned into the multiple cloning site. The sequence encoding maturebeta-lactamase is preceded by the leader peptide sequence of ompA toallow efficient secretion across the cytoplasmic membrane. Furthermore asequence encoding the first 12 amino acids (spacer sequence) of maturebeta-lactamase follows the ompA leader peptide sequence to avoid fusionof sequences immediately after the leader peptidase cleavage site, sincee.g. clusters of positive charged amino acids in this region woulddecrease or abolish translocation across the cytoplasmic membrane{Kajava, A. et al., 2000}. A SmaI restriction site serves for libraryinsertion. An upstream FseI site and a downstream NotI site, which wereused for recovery of the selected fragment, flank the SmaI site. Thethree restriction sites are inserted after the sequence encoding the 12amino acid spacer sequence in such a way that the bla gene istranscribed in the −1 reading frame resulting in a stop codon 15 bpafter the NotI site. A +1 bp insertion restores the bla ORF so thatbeta-lactamase protein is produced with a consequent gain of Ampicillinresistance.

The vector pMAL9.1 was constructed by cloning the lamB gene into themultiple cloning site of pEH1 {Hashemzadeh-Bonehi, L. et al., 1998}.Subsequently, a sequence was inserted in lamB after amino acid 154,containing the restriction sites FseI, SmaI and NotI. The reading framefor this insertion was constructed in such a way that transfer offrame-selected DNA fragments excised by digestion with FseI and NotIfrom plasmid pMAL4.31 yields a continuous reading frame of lamB and therespective insert.

The vector pHIE11 was constructed by cloning the fhuA gene into themultiple cloning site of pEH1. Thereafter, a sequence was inserted infhuA after amino acid 405, containing the restriction site FseI, XbaIand NotI. The reading frame for this insertion was chosen in a way thattransfer of frame-selected DNA fragments excised by digestion with FseIand NotI from plasmid pMAL4.31 yields a continuous reading frame of fhuAand the respective insert.

Cloning and evaluation of the library for frame selection. Genomic S.epidermidis DNA fragments were ligated into the SmaI site of the vectorpMAL4.31. Recombinant DNA was electroporated into DH10B electrocompetentE. coli cells (GIBCO BRL) and transformants plated on LB-agarsupplemented with Kanamycin (50 μg/ml) and Ampicillin (50 μg/ml). Plateswere incubated over night at 37° C. and colonies collected for largescale DNA extraction. A representative plate was stored and saved forcollecting colonies for colony PCR analysis and large-scale sequencing.A simple colony PCR assay was used to initially determine the roughfragment size distribution as well as insertion efficiency. Fromsequencing data the precise fragment size was evaluated, junctionintactness at the insertion site as well as the frame selection accuracy(3n+1 rule).

Cloning and evaluation of the library for bacterial surface display.Genomic DNA fragments were excised from the pMAL4.31 vector, containingthe S. epidermidis library with the restriction enzymes FseI and NotI.The entire population of fragments was then transferred into plasmidspMAL9.1 (LamB) or pHIE11 (FhuA), which have been digested with FseI andNotI.

Using these two restriction enzymes, which recognise an 8 bp GC richsequence, the reading frame that was selected in the pMAL4.31 vector ismaintained in each of the platform vectors. The plasmid library was thentransformed into E. coli DHSalpha cells by electroporation. Cells wereplated onto large LB-agar plates supplemented with 50 μg/ml Kanamycinand grown over night at 37° C. at a density yielding clearly visiblesingle colonies. Cells were then scraped off the surface of theseplates, washed with fresh LB medium and stored in aliquots for libraryscreening at −80° C.

Results

Libraries for frame selection. Two libraries (LSE-70 and LSE-150) weregenerated in the pMAL4.31 vector with sizes of approximately 70, 150 and300 bp, respectively. For each library, ligation and subsequenttransformation of approximately 1 μg of pMAL4.31 plasmid DNA and 50 ngof fragmented genomic S. epidermidis DNA yielded 4×105 to 2×106 clonesafter frame selection. To assess the randomness of the libraries,approximately 600 randomly chosen clones of LSE-70 were sequenced. Thebioinformatic analysis showed that of these clones only very few werepresent more than once. Furthermore, it was shown that 90% of the clonesfell in the size range between 16 and 61 bp with an average size of 34bp (FIG. 2). Almost all sequences followed the 3n+1 rule, showing thatall clones were properly frame selected.

Bacterial surface display libraries. The display of peptides on thesurface of E. coli required the transfer of the inserts from the LSElibraries from the frame selection vector pMAL4.31 to the displayplasmids pMAL9.1 (LamB) or pHIE11 (FhuA). Genomic DNA fragments wereexcised by FseI and NotI restriction and ligation of 5 ng inserts with0.1 μg plasmid DNA and subsequent transformation into DHSalpha cellsresulted in 2−5×106 clones. The clones were scraped off the LB platesand frozen without further amplification.

Example 3 Identification of highly immunogenic peptide sequences from S.epidermidis using bacterial surface displayed genomic libraries andhuman serum Experimental Procedures

MACS screening. Approximately 2.5×10⁸ cells from a given library weregrown in 5 ml LBmedium supplemented with 50 μg/ml Kanamycin for 2 h at37° C. Expression was induced by the addition of 1 mM IPTG for 30 min.Cells were washed twice with fresh LB medium and approximately 2×10⁷cells re-suspended in 100 μl LB medium and transferred to an Eppendorftube.

10 μg of biotinylated, human IgGs purified from serum was added to thecells and the suspension incubated over night at 4° C. with gentleshaking. 900 μl of LB medium was added, the suspension mixed andsubsequently centrifuged for 10 min at 6,000 rpm at 4° C. (For IgAscreens, 10 μg of purified IgAs were used and these captured withbiotinylated anti-human-IgG secondary antibodies). Cells were washedonce with 1 ml LB and then re-suspended in 100 μl LB medium. 10 μl ofMACS microbeads coupled to streptavidin (Miltenyi Biotech, Germany) wereadded and the incubation continued for 20 min at 4° C. Thereafter 900 μlof LB medium was added and the MACS microbead cell suspension was loadedonto the equilibrated MS column (Miltenyi Biotech, Germany) which wasfixed to the magnet. (The MS columns were equilibrated by washing oncewith 1 ml 70% EtOH and twice with 2 ml LB medium.)

The column was then washed three times with 3 ml LB medium. Afterremoval of the magnet, cells were eluted by washing with 2 ml LB medium.After washing the column with 3 ml LB medium, the 2 ml eluate was loadeda second time on the same column and the washing and elution processrepeated. The loading, washing and elution process was performed a thirdtime, resulting in a final eluate of 2 ml.

A second round of screening was performed as follows. The cells from thefinal eluate were collected by centrifugation and re-suspended in 1 mlLB medium supplemented with 50 μg/ml Kanamycin. The culture wasincubated at 37° C. for 90 min and then induced with 1 mM IPTG for 30min. Cells were subsequently collected, washed once with 1 ml LB mediumand suspended in 10 μl LB medium. Since the volume was reduced, 10 μg ofhuman, biotinylated IgGs was added and the suspension incubated overnight at 4° C. with gentle shaking. All further steps were exactly thesame as in the first selection round. Cells selected after two rounds ofselection were plated onto LB-agar plates supplemented with 50 μg/mlKanamycin and grown over night at 37° C.

Evaluation of selected clones by sequencing and Western blot analysis.Selected clones were grown over night at 37° C. in 3 ml LB mediumsupplemented with 50 μg/ml Kanamycin to prepare plasmid DNA usingstandard procedures. Sequencing was performed at MWG (Germany).

For Western blot analysis approximately 10 to 20 μg of total cellularprotein was separated by 10% SDS-PAGE and blotted onto HybondC membrane(Amersham Pharmacia Biotech, England). The LamB or FhuA fusion proteinswere detected using human serum as the primary antibody at a dilution ofapproximately 1:5,000 and anti-human IgG or IgA antibodies coupled toHRP at a dilution of 1:5,000 as secondary antibodies. Detection wasperformed using the ECL detection kit (Amersham Pharmacia Biotech,England). Alternatively, rabbit anti FhuA or mouse anti LamB antibodieswere used as primary antibodies in combination with the respectivesecondary antibodies coupled to HRP for the detection of the fusionproteins.

Results

Screening of bacterial surface display libraries by magnetic activatedcell sorting (MACS) using biotinylated Igs. The libraries LSE-70 inpMAL9.1 and LSE-150 in pHIE11 were screened with a pool of biotinylated,human IgG from patient sera (see Example 1: Preparation of antibodiesfrom human serum). In addition, a S. aureus library (LSA-300 in pHIE11)was also screened with the same serum pool, P15-IgG. The selectionprocedure was performed as described under Experimental procedures. FIG.3A shows a representative example of a screen with the LSE-70 libraryand P15-IgGs. As can be seen from the colony count after the firstselection cycle from MACS screening, the total number of cells recoveredat the end is drastically reduced from approximately 3×10⁷ cells to app.2×10⁴ cells, whereas the selection without antibodies added showed areduction to about 1×10⁴ cells (FIG. 3A). After the second round, asimilar number of cells was recovered with P15-IgG, while app. 8-foldfewer cells were recovered when no IgGs from human serum were added,clearly showing that selection was dependent on S. epidermidis specificantibodies. To evaluate the performance of the screen, 26 selectedclones were picked randomly and subjected to Western blot analysis withthe same, pooled serum (FIG. 3B). This analysis revealed that 70% of theselected clones showed reactivity with antibodies present in therelevant serum whereas the control strain expressing LamB without a S.epidermidis specific insert did not react with the same serum. Ingeneral, the rate of reactivity was observed to lie within the range of35 to 75%. Colony PCR analysis showed that all selected clones containedan insert in the expected size range.

Subsequent sequencing of a larger number of randomly picked clones (600to 1000 per screen) led to the identification of the gene and thecorresponding peptide or protein sequence that was specificallyrecognized by the human serum used for screening. The frequency withwhich a specific clone is selected reflects at least in part theabundance and/or affinity of the specific antibodies in the serum usedfor selection and recognizing the epitope presented by this clone. Table1 summarizes the data obtained for the three performed screens, butlists only those genes, which have not been identified by previousscreens. All clones that are presented in Table 1 have been verified byWestern blot analysis using whole cellular extracts from single clonesto show the indicated reactivity with the pool of human serum used inthe respective screen. As can be seen from Table 1, distinct regions ofthe identified ORF are identified as immunogenic, since variably sizedfragments of the proteins are displayed on the surface by the platformproteins. The screen with the S. aureus library revealed one novelantigen, which had not been identified in previous screens.

It is further worth noticing that most of the genes identified by thebacterial surface display screen encode proteins that are eitherattached to the surface of S. epidermidis and/or are secreted. This isin accordance with the expected role of surface attached or secretedproteins in virulence of S. epidermidis.

Example 4 Gene Distribution Studies with Highly Immunogenic ProteinsIdentified from S. epidermidis

Gene distribution of staphylococcal antigens by PCR. An ideal vaccineantigen would be an antigen that is present in all, or the vast majorityof strains of the target organism to which the vaccine is directed. Inorder to establish whether the genes encoding the identifiedStaphylococcus epidermidis antigens occur ubiquitously in S. epidermidisand coagulase negative Staphylococcus strains, PCR was performed on aseries of independent S. epidermidis and coagulase negativeStaphylococcus isolates with primers specific for the gene of interest.Oligonucleotide sequences as primers were designed for all identifiedORFs yielding products of approximately 1,000 bp, if possible coveringall identified immunogenic epitopes. Genomic DNA of all Staphylococcusstrains was prepared as described under Example 2. PCR was performed ina reaction volume of 25 μl using Taq polymerase (1U), 200 nM dNTPs, 10pMol of each oligonucleotide and the kit according to the manufacturersinstructions (Invitrogen, The Netherlands). As standard, 30 cycles (1×:5 min. 95° C., 30×: 30 sec. 95° C., 30 sec. 56° C., 30 sec. 72° C., 1× 4min. 72° C.) were performed, unless conditions had to be adapted forindividual primer pairs.

Results

Exemplarily, a number of genes encoding immunogenic proteins were testedby PCR for their presence in 42 different coagulase negativeStaphylococcus (CNS) or S. epidermidis strains. FIG. 4 shows the PCRreaction for ORF1163 with all indicated 42 strains. It was expected thatnot all of the CNS strains represent S. epidermidis isolates. Thereforeit was not surprising that 6 of the 31 CNS strains were negative for allgenes analysed. Some of the eight selected genes encoding identifiedantigens and analysed by PCR, were present in many strains tested (e.g.ORF0026, ORF0217 and ORF1163), rendering them as good candidates forfurther development. A few genes were present in only a smaller numberof the tested 42 strains (e.g. ORF0742 and ORF2700). This result mayindicate the absence of the gene in the analysed isolates, or it couldbe due to a variation in the sequence used for the oligonucleotide forthe PCR analysis. Interestingly, none of the eight analysed genes showedany variation in size. Sequencing of the generated PCR fragment from onestrain and subsequent comparison to the RP62A strain confirmed theamplification of the correct DNA fragment. Importantly, the identifiedantigens, which are well conserved in all strains in sequence and sizeconstitute novel vaccine candidates to prevent infections by S.epidermidis. As can be seen in Table 1, 20 of the listed 30 S.epidermidis antigens have a homolog in S. aureus COL with at least 50%sequence identity at the amino acid level, 4 have homologs with anidentity below 50% and 6 antigens do not possess a homologous sequencein S. aureus COL. This indicates that several of the antigens have alsothe potential to show cross-protection with other Staphylococcal strainssuch as S. aureus.

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TABLE 1 Immunogenic proteins identified by bacterial surface display.No. of selected Location of Gene Seq. S. epidermidis clones peridentified Homology dis- ID or aureus anti- Putative function ORF andimmunogenic with tribu- (DNA, genic protein (by homology) predictedimmunogenic aa* screen region (aa) S. aureus tion^(§) Prot.) ORF00026LPXTG-motif cell wall 6-28, 54-59, 135-147, 193-205, 274-279, A: 5396-449 32% 26/36  1, 32 anchor domain protein 284-291, 298-308,342-347, 360-366, SA2668 380-386, 408-425, 437-446, 457-464, 467-477,504-510, 517-530, 535-543, 547-553, 562-569, 573-579, 592-600, 602-613,626-631, 638-668 ORF00027 autolysin, putative 5-24, 101-108, 111-117,128-142, 170- A: 3  8-21 53% n.d.  2, 33 184, 205-211, 252-267, 308-316,329- SA2666 337, 345-353, 360-371, 375-389, 393- 399, 413-419, 429-439,446-456, 471- 485, 495-507, 541-556, 582-588, 592- 602, 607-617,622-628, 630-640 ORF00217 toxin resistance protein, 10-20, 23-33, 40-45,59-65, 72-107, 113- A: 2 33-59 66% 29/36  3, 34 putative 119, 127-136,151-161 SA2541 ORF00259 helicase-related protein 4-16, 28-34, 39-61,66-79, 100-113, 120- A: 2 913-933 65% n.d.  4, 35 127, 130-137, 142-148,150-157, 192- SA2499 201, 203-210, 228-239, 245-250, 256- 266, 268-278,288-294, 312-322, 336- 344, 346-358, 388-396, 399-413, 425- 430,445-461, 464-470, 476-482, 486- 492, 503-511, 520-527, 531-541, 551-558, 566-572, 609-625, 635-642, 650- 656, 683-689, 691-705, 734-741,750- 767, 782-789, 802-808, 812-818, 837- 844, 878-885, 907-917, 930-936ORF00545 tagatose 1,6- 5-12, 20-27, 46-78, 85-92, 104-112, 121- A: 10177-206 90% n.d.  5, 36 diphosphate aldolase 132, 150-167, 179-185,200-213, 221- SA2183 (lacD) 227, 240-264, 271-279, 282-290, 311- 317ORF00646 UDP-N- 18-24, 31-40, 45-51, 89-97, 100-123, A: 3 132-152 72%n.d.  6, 37 acetylglucosamine 2- 127-132, 139-153, 164-170, 184-194,SA2103 epimerase 200-205, 215-238, 244-255, 257-270, 62% 272-280,289-302, 312-318, 338-348, SA0151 356-367 ORF00742 M23/M37 peptidase7-16, 39-45, 73-83, 90-98, 118-124, 130- A: 14, B: 7 687-730 18%  5/36 7, 38 domain protein protein 136, 194-204, 269-280, 320-327, 373-SA0379 381, 389-397, 403-408, 424-430, 436- 441, 463-476, 487-499,507-514, 527- 534, 540-550, 571-577, 593-599, 620- 629, 641-647,650-664, 697-703, 708- 717, 729-742, 773-790, 794-805, 821- 828,830-837, 839-851, 858-908, 910- 917, 938-947, 965-980, 1025-1033,1050-1056, 1073-1081, 1084-1098, 1106-1120, 1132-1140, 1164-1170,1185-1194, 1201-1208, 1215-1224, 1226-1234, 1267-1279, 1325-1331,1356-1364, 1394-1411, 1426-1439, 1445-1461, 1498-1504, 1556-1561,1564-1573, 1613-1639, 1648-1655, 1694-1714, 1748-1755, 1778-1785,1808-1813, 1821-1827, 1829-1837, 1846-1852, 1859-1865, 1874-1883,1895-1900, 1908-1913, 1931-1937, 1964-1981, 1995-2005, 2020-2033,2040-2047, 2103-2109, 2118-2127, 2138-2144, 2166-2175, 2180-2187,2220-2225, 2237-2242, 2247-2253, 2273-2281, 2286-2306, 2314-2320,2323-2345, 2350-2355, 2371-2384, 2415-2424, 2426-2431, 2452-2472,2584-2589, 2610-2621, 2638-2655, 2664-2670, 2681-2690, 2692-2714,2724-2730 ORF00788 conserved hypothetical 10-40, 53-59, 79-85, 98-104,117-122, B: 1 254-292 none  4/36  8, 39 protein 130-136, 144-158,169-175, 180-185, 203-223, 232-237, 243-254, 295-301 ORF00891 celldivision protein 28-50, 67-85, 93-115, 120-134, 144-179, B: 5 275-316;378- 69% n.d.  9, 40 (42% FtsK (ftsK) 240-249, 328-340, 354-360,368-400, 401 SA1295 ORF01770) 402-417, 419-427, 429-445, 447-455, 42%463-468, 472-480, 485-500, 502-510, SA1791 512-534, 537-546, 553-558,582-594, 619-637, 645-654, 690-709, 735-745, 749-756, 786-792 ORF00894metalloprotease, 5-16, 21-30, 33-40, 52-74, 101-108, 116- A: 1 191-20876% n.d. 10, 41 insulinase family, 122, 164-182, 185-219, 256-261, 273-SA1298 putative 279, 285-291, 297-304, 312-328, 331- 338, 355-362,364-371, 373-401, 411- 423 ORF00988 membrane-bound 34-55, 67-74, 85-93,105-115, 138-152, A: 1 216-231 74% n.d. 11, 42 protein LytR 161-171,182-189, 197-205, 213-219, SA1398 232-239, 241-248, 250-263, 272-277,288-299 ORF01054 ABC transporter, ATP- 21-27, 32-37, 43-51, 67-74,82-92, 94- B: 4 214-280 75% n.d. 12, 43 (31% binding protein 100,106-112, 140-149, 153-159, 164- SA0779 ORF00724) 182, 193-215, 222-227,260-267, 308- 28% 322, 330-340, 378-387, 396-403, 417- SA2036 432,435-441, 448-465, 476-482, 488- 498, 500-510 ORF01163 lipoprotein YaeC,4-21, 29-52, 80-87, 104-123, 126-133, A: 3, B: 8  33-108 79% 31/36 13,44 (38% putative 141-157, 182-189, 194-202, 214-220, SA0884 ORF02440)227-235, 242-252 35% SA0506 ORF01182 UDP-sugar hydrolase, 12-18, 20-27,29-59, 64-72, 84-90, 96- A: 3 76-96 71% n.d. 14, 45 putative 103,109-121, 125-155, 164-177, 179- SA0926 186, 188-201, 216-227, 235-253,259- 274, 276-294, 296-310, 322-339, 341- 348, 369-379, 398-403, 409-421ORF01515 hypothetical protein 4-15, 24-41, 71-80, 104-111, 113-119, A:17  4-45 none  5/36 15, 46 123-130, 139-149, 168-178, 187-200 ORF01596conserved hypothetical 13-19, 32-37, 44-56 A: 3  1-14 60% n.d. 16, 47protein SA1972 ORF01755 Mrp protein 6-11, 16-35, 75-81, 95-100, 126-139,A: 2, B: 8 213-276; 579- 31% n.d. 17, 48 206-214, 225-233, 241-259,268-276, 621; 1516- SA1806 319-325, 339-360, 371-401, 435-441, 1559 28%452-459, 462-472, 491-503, 505-516, SA2150 549-556, 567-580, 590-595,612-622, 624-630, 642-648, 656-662, 687-693, 698-704, 706-712, 736-750,768-777, 784-789, 812-818, 847-858, 894-900, 922-931, 938-949, 967-984,986-992, 1027-1032, 1041-1054, 1082-1088, 1091-1097, 1119-1124,1234-1240, 1250-1258, 1274-1289, 1299-1305, 1392-1398, 1400-1405,1429-1442, 1460-1474, 1505-1514, 1531-1537, 1540-1552, 1558-1571,1582-1587, 1616-1623, 1659-1666, 1671-1677, 1680-1686, 1698-1704,1706-1712, 1768-1774, 1783-1797, 1814-1819, 1849-1855, 1870-1876,1890-1897, 1947-1953, 1972-1980, 1999-2013, 2044-2051, 2068-2084,2093-2099, 2122-2131, 2142-2147, 2156-2163, 2170-2179, 2214-2220,2235-2245, 2271-2281, 2287-2293, 2308-2317, 2352-2362, 2373-2378,2387-2407, 2442-2448, 2458-2474, 2507-2516, 2531-2537, 2540-2551,2555-2561, 2586-2599, 2617-2627, 2644-2649, 2661-2675, 2685-2692,2695-2707, 2733-2739, 2741-2747, 2774-2783, 2788-2795, 2860-2870,2891-2903, 2938-2947, 2973-2980, 2993-2999, 3004-3030, 3046-3059,3066-3077, 3082-3088, 3120-3132, 3144-3149, 3153-3169, 3200-3212,3232-3256, 3276-3290, 3308-3322, 3330-3338, 3353-3360, 3363-3371,3390-3408, 3431-3447, 3454-3484, 3503-3515, 3524-3541, 3543-3550,3560-3567, 3586-3599, 3616-3621, 3642-3647, 3663-3679 ORF02009 2-oxoacid 19-41, 43-49, 55-62, 67-74, 114-121, B: 4 12-56 64% n.d. 18, 49(32% dehydrogenase, 130-140, 188-197, 208-217, 226-232, SA1560 ORF01373& E2 component, 265-287, 292-299, 301-319, 372-394, 32% ORF01042)lipoamide 400-410, 421-427 SA1104 31% SA1448 ORF02025integrase/recombinase 6-12, 44-51, 53-60, 67-88, 91-100, 104- B: 3 60-138 85% n.d. 19, 50 (35% XerD (xerD) 123, 137-142, 148-158, 161-168,175- SA1540 ORF00861) 201, 204-210, 222-231, 239-253, 258- 35% 264,272-282 SA1269 ORF02209 NADH dehydrogenase, 4-63, 69-104, 110-121,124-131, 134- A: 2 509-528 66% n.d. 20, 51 (37% putative 152, 161-187,204-221, 223-237, 239- SA0679 ORF01212) 296, 298-310, 331-365, 380-405,423- 38% 451, 470-552, 554-562, 574-581, 592- SA0955 649, 651-658,661-671, 673-707, 713- 734, 741-748, 758-765, 773-790 ORF02289fibrinogen-binding 89-94, 102-115, 123-129, 181-188, 200- B: 2 213-26541% n.d. 21, 52 protein SdrG 206, 211-235, 239-249, 267-281, 295- SA0610310, 316-321, 331-341, 344-359, 365- 32% 386, 409-422, 443-453, 495-506,514- SA0608 521, 539-547, 553-560, 563-570, 586- 596, 621-626, 633-638,651-657, 666- 30% 683, 697-705, 731-739, 761-768, 865- SA0609 883ORF02329 glutamyl-tRNA 5-20, 24-34, 37-43, 92-102, 134-139, A: 7 145-18382% n.d. 22, 53 synthetase (gltX) 156-162, 184-191, 193-205, 207-213,SA0574 225-231, 241-247, 259-267, 269-286, 337-350, 365-372, 378-386,399-413, 415-421, 447-457, 467-481 ORF02393 dimethyladenosine 12-19,29-41, 43-57, 80-98, 106-141, A: 3, B: 2 237-287 85% n.d. 23, 54transferase (ksgA) 143-156, 172-183, 185-210, 214-220, SA0536 226-234,278-287 ORF02412 conserved hypothetical 5-12, 32-48, 50-72, 75-81, 88-94A: 1, B: 1 16-40 none n.d. 24, 55 (100% protein ORF02349 & ORF01658 &ORF00589 & ORF00701 ORF02680 Metallo-beta-lactamase 4-21, 29-42, 48-62,65-80, 95-101, 103- A: 22 208-230 98% 20/36 25, 56 (74% superfamilydomain 118, 122-130, 134-140, 143-152, 155- SA0046 ORF02594) protein165, 182-192, 198-208, 232-247, 260- 73% 268, 318-348, 364-369, 380-391,403- SA0064 411, 413-424 ORF02700 hypothetical protein 4-18, 65-75,82-92, 123-140, 144-159, A: 1 174-195 none  2/36 26, 57 (lipoprotein)166-172, 188-194 ORF02825 malate: quinone 7-20, 58-71, 94-101, 110-119,199-209, B: 2 310-350 83% n.d. 27, 58 (83% oxidoreductase 231-242,247-254, 267-277, 282-290, SA2623 ORF00132, 297-306, 313-319, 333-342,344-369, 49% 67% 390-402, 414-431, 436-448, 462-471 SA2362 ORF02706, 51%ORF00369) ORF02853 hypothetical protein 4-25, 37-44, 53-59, 72-78,86-99, 119- A: 1 308-331 61% n.d. 28, 59 128, 197-203, 209-218, 220-226,233- SA0129 244, 246-254, 264-271, 277-289, 407- 430, 437-445, 464-472,482-488, 503- 509 CRF0299 Hypothetical protein 4-12, 14-43, 52-58 A: 3,B: 4 43-58 none n.d. 29, 60 CRF1769 Hypothetical protein 4-14, 21-29,35-49 A: 6 38-50 none n.d. 30, 61 SA1169 fibrinogen-binding 4-19, 31-37,58-72, 94-108 C: 2  1-72 none n.d. 31, 62 protein precursor-relatedprotein

1. An isolated hyperimmune serum-reactive antigen consisting of afragment of SEQ ID NO: 52, wherein said fragment comprises an amino acidsequence selected from the group consisting of amino acids 89-94,102-115, 123-129, 181-188, 200-206, 211-235, 239-249, 267-281, 295-310,316-321, 331-341, 344-359, 365-386, 409-422, 443-453, 495-506, 514-521,539-547, 553-560, 563-570, 586-596, 621-626, 633-638, 651-657, 666-683,697-705, 731-739, 761-768, 865-883 and 213-265 of SEQ ID NO: 52, andwherein said fragment is less than 892 amino acids in length.
 2. Animmunogenic composition comprising the isolated hyperimmuneserum-reactive antigen of claim
 1. 3. The immunogenic composition ofclaim 2, comprising at least 2 different hyperimmune serum-reactiveantigens.
 4. The immunogenic composition of claim 2, further comprisingan adjuvant.
 5. The immunogenic composition of claim 3, furthercomprising an adjuvant.
 6. A fusion protein comprising the hyperimmuneserum-reactive antigen according to claim
 1. 7. An immunogeniccomposition comprising the fusion protein of claim
 6. 8. The immunogeniccomposition of claim 7, further comprising an adjuvant.
 9. A method ofinducing an immune response in a subject comprising: administering theimmunogenic composition of claim 2 to a subject; wherein an immuneresponse is induced in the subject.
 10. The method of claim 9, whereinthe subject is a human.
 11. The method of claim 9, wherein the subjecthas an S. epidermidis infection.
 12. A method of inducing an immuneresponse in a subject comprising: administering the immunogeniccomposition of claim 7 to a subject; wherein an immune response isinduced in the subject.
 13. The method of claim 12, wherein the subjectis a human.
 14. The method of claim 12, wherein the subject has an S.epidermidis infection.